Broadly reactive mosaic peptide for influenza vaccine

ABSTRACT

The invention provides for mosaic influenza virus HA and NA sequences and uses thereof.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of the filing date of U.S.application Ser. No. 61/785,071, field on Mar. 14, 2013, the disclosureof which is incorporated by reference herein.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with government support under 2008-55620-19132awarded by the USDA/NIFA. The government has certain rights in theinvention.

BACKGROUND

Influenza viruses are a significant health concern for animals andhumans. The World Health Organization (WHO) estimates that every yearinfluenza virus infects up to 1 billion people, with 3-5 million casesof severe disease and 300,000-500,000 deaths annually (Meltzer et al.,1999). The traditional approach to controlling influenza A virus isbased on diagnosis, treatment and prevention through vaccination. Eachof these approaches, however, has flaws (e.g., antiviral resistance,incomplete protection, and improper vaccine distribution), e.g.,treating every case with antiviral drugs is not a viable option becauseit is often ineffective and leads to viral resistance.

Highly pathogenic avian influenza (HPAI) H5N1 viruses have spread as faras Eurasia and Africa since their first emergence in 1996. These virusesinfect a range of domestic and wild avian species as well as mammals(Pollack et al., 1998; Lazzari and Stohr, 2004), and pose a pandemicthreat (Allen, 2006; Anonymous, 2005; Conly and Johmston, 2004). Currentprevention and treatment strategies for H5N1 virus are antiviral,vaccine-based, or involve non-pharmaceutical measures, such as patientisolation or hand sanitation (Alexander et al., 2007; Ferguson et al.,2005; Ferguson et al., 2006; Iwami et al., 2008; Lipsitch et al., 2007;Stilanakis et al., 1998). However, these approaches have flaws (Iwami etal., 2008; Lipsitch et al., 2007; Lipsitch et al., 2009; Gandon et al.,2001).

Generation of inactivated vaccines (INV) has been optimized for seasonalflu, but presents several challenges for H5N1 viruses, including: 1)continual evolution of the viruses makes predicting a vaccine straindifficult; 2) egg propagation of vaccine stock is hindered due to thehigh lethality of H5N1 viruses to eggs and the poultry that providethem; and 3) the six to nine month time-period required to produce INVmay be too long to protect large populations during a pandemic. Inaddition, initial studies in mice, ferrets and phase 1 human clinicaltrials have demonstrated that INV and other split-virion vaccines mayrequire higher doses of antigen than traditional INV, with more than oneadministration needed to provide protective immunity (Cox et al., 2004;Ehrlich et al., 2008; Wright, 2008). Live vaccines elicit both humoraland cellular immune responses. However, they are not recommended ininfants, elderly, or immuno-compromised individuals because they cancause pathogenic reactions (Jefferson et al., 2005; Kunisaki and Janoff,2009; Mostow et al., 1969; Peck, 1968). Moreover, live vaccines canrevert to wild-type viruses, potentially leading to vaccine failure anddisease outbreaks (Mostow et al., 1979).

Current vaccines need to be improved to overcome limited crossprotection, short duration of immunity and/or lack of robust protection.For instance, a critical failure in preparation for influenza pandemicsand seasonal epidemics is the absence of a universal vaccine. This isdue in part to the extraordinary genetic and antigenic variation of thevirus, a consequence of rapid evolution in the form of antigenic driftand shift. Indeed, influenza strains vary by 1-2% per year, and vaccinesgenerally do not elicit protection from one year to the next,necessitating frequent vaccine updates. This diversity represents asignificant challenge to the development of a broadly effective vaccine,as no single viral variant can induce immunity across observed fieldstrains, and incorporating all circulating variants into one multivalentvaccine isn't feasible.

Multiple approaches have been studied to develop a universal influenzavaccine that could be applied to H5N1 viruses. One approach is to useconserved sequences such as the stalk region of HA or the internal NP orM1 proteins. Another approach involves consensus sequences that combinemany H5N1 hemagglutinin sequences into a single gene. Of theseapproaches, only the consensus approach has shown partial protectionagainst a diverse panel of H5N1 isolates. Nevertheless, a broadlyeffective strategy for H5N1, or other pandemic viruses, control remainselusive.

SUMMARY OF THE INVENTION

A mosaic influenza virus sequence is generated in silico from naturalsequences with an emphasis on current strains and is optimized formaximum T cell epitope coverage (e.g., maintaining contiguous epitopesequences) rather than on consensus residues. A mosaic sequence having alinear string of primarily natural occurring influenza virus T cellepitopes, optionally including B cell epitopes and/or T cytotoxiclymphocyte (TCL) epitopes, would likely provide robust and broadprotection against challenge. That is because an objective scoringmechanism is employed that optimizes for maximum T cell epitope coverageof the known diversity of wild-type influenza. Consequently, thesynthetic protein that is generated is less subject to the inherentbiases in the body of publically-available data. Moreover, the mosaicsequence is more likely to be functional and properly folded.

As described below, a modified vaccinia Ankara (MVA) vector was used toexpress a mosaic H5 HA gene (H5M). The MVA vector offers severaladvantages such as 1) safety, 2) stability, 3) rapid induction ofhumoral and cellular responses, and 4) multiple routes of inoculation.In mice, a single dose of MVA-H5M construct provided sterilizingimmunity (no detectable virus in lung tissues post challenge) againstH5N1 HPAI clades 0, 1 and 2 viruses. Furthermore, MVA-H5M provided fullprotection as early as 10 days post exposure and as long as 6 monthspost-vaccination. Both neutralizing antibodies and antigen-specific CD4⁺and CD8⁺ T cells were detected at 5 months post-vaccination. Inaddition, MVA-H5M also provided cross subtype protection against H1N1virus (PR8) challenged. These results indicate that the mosaic vaccineapproach has great potential for broadening the efficacy of influenzavaccines, perhaps including protection against all influenza subtypes.

The invention thus provides a universal influenza vaccine with a mosaic(synthetic) antigen. In one embodiment, the invention provides anisolated polynucleotide comprising a nucleic acid segment for aninfluenza virus HA having SEQ ID NO:1 or a polypeptide having at least95%, e.g., at least 99%, amino acid sequence identity thereto, aninfluenza virus HA having SEQ ID NO:2 or a polypeptide having at least95%, e.g., at least 99%, amino acid sequence identity thereto, aninfluenza virus HA having SEQ ID NO:3 or a polypeptide having at least95%, e.g., at least 99%, amino acid sequence identity thereto, aninfluenza virus HA having SEQ ID NO:4 or a polypeptide having at least95%, e.g., at least 99%, amino acid sequence identity thereto, aninfluenza virus HA having SEQ ID NO:5 or a polypeptide having at least95%, e.g., at least 99%, amino acid sequence identity thereto, aninfluenza virus HA having SEQ ID NO:6 or a polypeptide having at least95%, e.g., at least 99%, amino acid sequence identity thereto, aninfluenza virus HA having SEQ ID NO:7 or a polypeptide having at least95%, e.g., at least 99%, amino acid sequence identity thereto, aninfluenza virus HA having SEQ ID NO:8 or a polypeptide having at least95%, e.g., at least 99%, amino acid sequence identity thereto, aninfluenza virus HA having SEQ ID NO:9 or a polypeptide having at least95%, e.g., at least 99%, amino acid sequence identity thereto, aninfluenza virus HA having SEQ ID NO:10 or a polypeptide having at least95%, e.g., at least 99%, amino acid sequence identity thereto, aninfluenza virus HA having SEQ ID NO:11 or a polypeptide having at least95%, e.g., at least 99%, amino acid sequence identity thereto, aninfluenza virus NA having SEQ ID NO:12 or a polypeptide having at least95%, e.g., at least 99%, amino acid sequence identity thereto, aninfluenza virus NA having SEQ ID NO:13 or a polypeptide having at least95%, e.g., at least 99%, amino acid sequence identity thereto, or aninfluenza virus NA having SEQ ID NO:14 or a polypeptide having at least95%, e.g., at least 99%, amino acid sequence identity thereto, or thecomplement of the nucleic acid segment, which polypeptide isimmunogenic, e.g., providing subtype protection against two or moredistinct viruses, e.g., from different clades (cross clade is two ormore clades). Sequences included are those with one or a few amino acidinsertions, so long as the resulting sequences result in theimmunogenicity, e.g., providing subtype protection against two or moredistinct viruses, e.g., from different clades. In one embodiment, thenucleic acid segment is operably linked to a promoter and/or atranscription termination sequence.

To generate the synthetic antigens of the invention, which includeepitopes representing a large number of primary influenza virusisolates, e.g., from circulating strains, influenza HA subtype and NAsubtype sequences were compiled. For example, a sequence for a mosaic H5antigen was generated in silico using over two thousand publishedinfluenza virus H5 sequences from the Influenza Research Database thatrepresent (nonduplicated) sequences of primary isolates and/orcirculating isolates. In one embodiment, the sequences representcirculating viruses from clades 1, 2.1.3, 2.2, 2.2.1, 2.3.2, 2.3.4and/or 7, or from clades 0 1, 2.1.1, 2.1.2, 2.1.3, 2.2, 2.2.1, 2.3.1,2.3.2, 2.3.3 2.3.4, 2.4, 2.6, 3, 4, 5, 6, 7, 8 and/or 9. The efficacy ofone of the generated sequences (SEQ ID NO:1) was tested in a highlypathogenic avian influenza (HPAI) model in mice using a recombinantpoxvirus (the attenuated MVA vector). The mosaic H5 (H5M) sequence wasfound to be quite effective and provided broad protection againstviruses from different clades, including protection against Clade 0,Clade 1, and Clade 2 viruses. Thus, a mosaic influenza virus antigen maybe employed as a vaccine that is administered as isolated protein,isolated nucleic acid or via a delivery vehicle, including a viralvector or virus like particle. The viral vector may be a heterologousviral vector, e.g., a vector from poxvirus, avipoxviruses such asfowlpox (FPV) or canarypox viruses, Newcastle Disease virus, adenovirus,alphaviruses, or other viruses, or an influenza virus such as a liveattenuated influenza virus. The present invention thus relates to newinfluenza vaccine constructs, and methods of making and using thoseconstructs.

In one embodiment, to generate a mosaic sequence, a genetic algorithm isemployed to generate, select and recombine in silico potential T-cellepitopes and/or B-cell epitopes, into “mosaic” protein sequences thatare antigenic and can provide greater coverage of global viral variantsthan any single wild-type protein. T-cell epitopes are generally fromabout 8 to about 15 amino acid residues in length, and B cell epitopesare generally from about 12 up to about 35 amino acid residues inlength. The combination of epitopes in a full length mosaic HA or NAsequence may be employed in nucleic acid vectors for administration orfor protein expression, or a fragment of the sequence which isimmunogenic may also be employed. An “immunogenic portion” of a fulllength sequence may be as few as 8 amino acids in length and up to oneor more residues shorter than a full length polypeptide, e.g., a fulllength HA-1. For instance, an immunogenic portion of a polypeptide is apolypeptide that is about 50%, 60%, 70%, 80%, 85%, 90%, 95% or 99% ofthe length of a corresponding full length polypeptide, such as a fulllength HA or HA-1, or NA, and elicits an immunogenic response that is atleast 30%, 40%, 50%, or more, of the immunogenic response of thecorresponding full-length polypeptide. As described herein, mosaicsequences may include characteristic residues at one or more positions,and in one embodiment, immunogenic portions of the mosaic sequences alsohave those characteristic residue(s).

The genetic algorithm based approach to mosaic sequence generation hasbeen employed with other HA A subtypes, e.g., H1, H3, H7, H9, and H10,HA B, and other influenza virus proteins, e.g., NA subtypes N1, N2 andN7. Because the approach incorporates a plurality of influenza epitopesinto a mosaic sequence, the resulting sequences are likely useful in asubtype specific universal influenza vaccine that can provide bothdomesticated animals and humans, and/or avians, the maximum possibleprotection against this devastating respiratory disease.

Thus, the invention provides a composition comprising a recombinantnucleic acid molecule having a nucleotide sequence, e.g., in a viralvector such as live recombinant poxvirus, that encodes a mosaicinfluenza virus antigen as described herein. In one embodiment, theviral vector genome comprises at least one expression cassette having apromoter operably linked to a heterologous open reading frame comprisingthe nucleotide sequence that elicits neutralizing antibodies and/or acytotoxic T cell response. In one embodiment, the composition includesmore than one nucleotide sequence, each encoding a different antigen, atleast one of which is a mosaic antigen, e.g., a mosaic HA or NApolypeptide. For example, the composition may include more than one liverecombinant poxvirus, e.g., different isolates having different antigensor one virus encoding more than one influenza virus antigen. Once therecombinant virus infects cells of a host animal, the antigen(s) isexpressed in an amount effective to induce an immune response. In oneembodiment, a live recombinant virus may be obtained from a culture ofisolated mammalian cells transfected or transformed with a recombinantvirus genome comprising the at least one expression cassette. Any cell,e.g., any avian or mammalian cell, such as a human, canine, bovine,equine, feline, swine, ovine, mink, or non-human primate cell, includingmutant cells, which supports efficient replication of virus can beemployed to isolate and/or propagate the viruses. In another embodiment,host cells are continuous mammalian or avian cell lines or cell strains.Viral vectors useful in the invention include but are not limited torecombinant adenovirus, retrovirus, lentivirus, herpesvirus, poxvirus,papilloma virus, or adeno-associated virus. Viral and non-viral vectorsmay be present in liposomes, e.g., neutral or cationic liposomes, suchas DOSPA/DOPE, DOGS/DOPE or DMRIE/DOPE liposomes, and/or associated withother molecules such as DNA-anti-DNA antibody-cationic lipid(DOTMA/DOPE) complexes.

The recombinant nucleic acid, viral vector or mosaic protein of theinvention may be administered via any route including, but not limitedto, intramuscular, subcutaneous, intranasal, buccal, rectal, intravenousor intracoronary administration, and transfer to host cells may beenhanced using electroporation and/or iontophoresis.

In one embodiment, a composition of the invention, such as a vaccine,e.g., for in ovo, mucosal, or parenteral administration, having arecombinant virus may include doses ranging from 1×10⁴ to 1×10⁸ plaqueforming units (PFU) or TCID₅₀, e.g., from 1×10⁴ to 1×10⁸ PFU or TCID₅₀,which may be administered as a single dose or in two or more doses, oreach dose may include from 1×10⁴ to 1×10⁸ PFU or TCID₅₀, e.g., from1×10⁴ to 1×10⁸ PFU or TCID₅₀, of recombinant virus, such as poxvirus.For instance, each dose may have the same number of PFU or TCID₅₀, orthe booster dose(s) may have higher or lower amounts relative to theinitial (priming) dose. The priming dose and/or booster dose(s) mayinclude an adjuvant. Additionally, the vector used for prime and boostmay be different. For example, a pox virus expressing the mosaic antigenmay be used for a primary dose, while another viral vector, DNA vector,RNA vector, or protein is used for the secondary dose, or vice versa.

In one embodiment, a composition of the invention encodes or comprisesan influenza virus HA and/or NA, which may induce a humoral response, acellular response, or both, and so likely provides cross-protection. Inone embodiment, the vaccine confers from 50 to 100% protection againstheterologous challenge (cross protection). In one embodiment, theadministration of a composition of the invention to avians or mammalsprovides for enhanced survival, e.g., after exposure to influenza virus,including survival rates of at least 35% or greater, for instance,survival rates of 50%, 60%, 70%, 75%, 80%, 85%, 90% or greater, relativeto survival rates in the absence of the administration of thatcomposition or any other prophylactic or therapeutic agent. Thecompositions of the invention are useful prophylactically ortherapeutically, e.g., against seasonal flu.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Schematic mechanisms for immune response evasion by influenzavirus. AR, TC, EEM are abbreviations for amino acids.

FIG. 2. Exemplary mosaic vaccine approach.

FIG. 3. Exemplary mosaic (synthetic) H5 protein sequence (SEQ ID NO:1).The mosaic H5N1 hemagglutinin (H5M) sequence was deduced from 2, 145 HAsequences. Lines above the sequence indicate known T-helper cell (blue),B-cell (green) or TCL (Red) epitopes that were found in the H5M(Influenza research database (IRD)).

FIG. 4. Selection of divergent challenge stains.

FIG. 5. MVA-H5M vaccine expresses higher level of protein than MVAexpressing wild type hemaggutinin (MVA-HA) and elicits broadneutralizing antibodies against avian influenza viruses. (A) Westernblot analysis of MVA-H5M, MVA-HA and MVA-LUC (negative control) infectedCEF cell lysates. HA from MVA-H5M was expressed as a cleavable proteinas same as HA wildtype of MVA-HA. The sizes of HA0, HA1 and HA2 are 75kDa, 50 kDa and 25 kDa, respectively. (B) Neutralizing antibody titersof vaccinated mice were measured at 4 week post-vaccination againstinfluenza A/VN/1203/04, A/MG/244/05, A/HK/483/97, A/Egypt/1/08, PR8(H1N1) or A/Aichi/2/1968 (H3N2) virus.

FIG. 6. MVA-H5M elicits broader epitope coverage than wild typehemagglutinin based vaccine. IFN-γ-producing CD4⁺ T cells from mice thatwere vaccinated with MVA-H5M or MVA-HA. Splenocytes from vaccinated micewere collected and stimulated with HA peptide pools from H5N1 viruses(as indicated with p1-p11). *P<0.05 or **P<0.01, Student's T test.

FIG. 7. Results of microneutralization assay against VN/1203/04,MONG/244/05 and Hong Kong/483/97 in mice immunized with H5M, inactivatedvirus or vector (MVA) alone.

FIG. 8. Neutralization titers comparison between MVA-H5M and MVA-HAvaccines against H5N1 viruses.

FIG. 9. MVA-H5M elicits T cells responses against PR8HA peptides.IFN-γ-producing CD8⁺ T cells from mice that were vaccinated with MVA-H5Mor MVA-HA vaccine. Splenocytes from vaccinated mice were collected andstimulated with HA peptide pools from H1N1 viruses (as indicated withp1-p9). *P<0.05, Student's T test.

FIG. 10. Survival over time in mice immunized with H5M, inactivatedvirus or vector (MVA) alone and challenged with VN/1203/04 (A);MONG/244/05(B); and HK/483/97 (C).

FIG. 11. Percent weight loss over time in mice immunized with H5M,inactivated virus or vector (MVA) alone and challenged with VN/1203/04(A); MONG/244/05 (B); and HK/483/97 (C).

FIG. 12. Lung viral titers in mice immunized with H5M, inactivated virusor vector (MVA) alone, for VN/1203/04 (A); MONG/244/05 (B); andHK/483/97 (C).

FIG. 13. MVA-H5M provides broad protection against multiple clades ofavian influenza virus, and H1N1 virus. Vaccine efficacies of a singledose of MVA-H5M or MVA-LUC against highly pathogenic avian influenzaviruses (A-G, J and M) and seasonal influenza viruses (H-I, K-L and N).Vaccinated mice were challenged at week 5 post-vaccination, and survivaldata were monitored for 14 days (n=8 per group).

FIG. 14. MVA-H5M provides short- and long-term immunities. (A-B) BALB/cmice were immunized with single dose MVA-H5M or MVA-LUC. 10 days or 6months post-vaccination, mice were challenged with a lethal dose ofinfluenza A/HK/483/97. (C) Neutralization titers from vaccinated mice at10 days and 6 months post-vaccination. (D) CD4+ and CD8+ T cellsresponses at 5 months post-vaccination.

FIG. 15. Histopathology in mice immunized with H5M, inactivated virus orvector (MVA) alone and challenged with VN/1203/04 (A); MONG/244/05 (B);and HK/483/97 (C).

FIG. 16. MVA-H5M reduces lung pathology and prevents viral replicationin the lung after challenged with avian influenza viruses. Lungs of micevaccinated with MVA-H5M (A, E, I, M, C, G, K and O) or MVA-LUC (B, F, J,N, D, H, L and P), challenged with A/VN/1203/04 (A-D), A/MG/244/05(E-H), A/HK/483/97 virus (I-L) or A/Egypt/1/08 (M-P). MVA-H5M vaccinatedmice showed normal to mild lung lesions compared to MVA-LUC-vaccinatedmice, which showed severe lung lesions including lung consolidation,WBCs infiltration, thickening of alveolar septa and alveolar edema.Lungs from mice that were administered MVA-H5M (C, G, K and O) orMVA-LUC (D, H, L and P) were processed by immunohistochemistry with H5N1specific antibody. Brown staining for viral antigen is indicated witharrow heads.

FIG. 17. Other exemplary mosaic influenza antigen sequences HIM (SEQ IDNO:2), HIM (SEQ ID NO:3), HIM (SEQ ID NO:4), HIM (SEQ ID NO:5), H2M (SEQID NO:6), H3M (SEQ ID NO:7), H7M (SEQ ID NO:8), H9M (SEQ ID NO:9), H10M(SEQ ID NO:10), HBM (SEQ ID NO:11), N1 (SEQ ID NO:12), N2 (SEQ IDNO:13), and N7 (SEQ ID NO:14).

FIG. 18. Conserved motifs in some of the sequences shown in FIG. 11.With regard to sequences that are related but include one or moresubstitutions to those sequences, those substitutions may be in theconserved regions but generally not in any signature residue.

FIG. 19. Codon usage tables for exemplary organisms.

DETAILED DESCRIPTION Definitions

As used herein, the term “isolated” refers to in vitro preparationand/or isolation of a nucleic acid molecule, e.g., vector or plasmid,peptide or polypeptide (protein), or virus of the invention, so that itis not associated with in vivo substances, or is substantially purifiedfrom in vitro substances. An isolated virus preparation is generallyobtained by in vitro culture and propagation, and is substantially freefrom other infectious agents.

A “recombinant” virus is one which has been manipulated in vitro, e.g.,using recombinant DNA techniques, to introduce changes to the viralgenome.

As used herein, the term “recombinant nucleic acid” or “recombinant DNAsequence or segment” refers to a nucleic acid, e.g., to DNA, that hasbeen derived or isolated from a source, that may be subsequentlychemically altered in vitro, so that its sequence is not naturallyoccurring, or corresponds to naturally occurring sequences that are notpositioned as they would be positioned in the native genome. An exampleof DNA “derived” from a source, would be a DNA sequence that isidentified as a useful fragment, and which is then chemicallysynthesized in essentially pure form. An example of such DNA “isolated”from a source would be a useful DNA sequence that is excised or removedfrom said source by chemical means, e.g., by the use of restrictionendonucleases, so that it can be further manipulated, e.g., amplified,for use in the invention, by the methodology of genetic engineering.

Conserved or Consensus Influenza Virus Sequences Versus Mosaic InfluenzaVirus Sequences

In an effort to develop vaccines that maximize the representation ofantigenic features present in diverse vial populations, a series ofstrategies have been proposed. The approaches have includedconcatenating commonly recognized T-cell epitopes (Palker et al., 1989),creating psuedoprotein strings of T-cell epitopes (De Groot et al.,2005) and generating consensus overlapping peptide sets from proteins(Thomson et al., 2005). Evolutionary approaches such as the use ofconsensus sequences (Gao et al., 2004, 2005; Gaschen et al., 2002), andthe most recent common ancestor (MRCA) of viral populations, have alsobeen proposed with the assumption that these approaches capture viraldiversity (Gaschen et al., 2002). Unfortunately, experimental studies inanimal models using these strategies have documented underwhelminghumoral immune responses (Doria-Rose et al., 2005; Gao et al., 2005).

Because of antigenic drift of influenza viruses, the components of aninfluenza virus vaccine are tailored annually to match the strains thatwould most likely be dominant in the population for the upcominginfluenza season. Yearly vaccinations are required because each seasonalvaccine elicits neutralizing antibodies that are specific only for thevaccine strains and closely related isolates. In case of a new pandemicstrain, it takes several months to reformulate the vaccine that matchesto the new strain.

To overcome these problems and to stop the spread of influenzaindefinitely, a single broadly protective vaccine or universal vaccineis needed. Thus, conserved influenza virus sequences from differentstrains, or consensus sequences, have been employed to provide anantigen with broad protective properties. For example, conservedinfluenza virus proteins include NP and M1, which are targets forcellular immunity. There is a certain immunogenic region of M1 (M58-66:GILGFVFTL) that is evolutionarily conserved (Thomas et al., 2006) and is100% conserved in almost all the strains of influenza virus includingH1N1, H5N1, H3N2 and pandemic H1N1, and the extracellular N-terminaldomain of M2 protein (eM2), a 23 amino acid peptide, is highly conservedin all human influenza A strains. Universal neutralizing antibodies havebeen isolated against the conserved HA2 region of hemagglutinin (HA)(Ekiert et al., 2009; Steel et al., 2010). However, universalneutralizing antibodies are rare, have low affinity, and cannot beinduced in large quantities during infection or vaccination.

Sequence alignments are relied on to yield a “consensus” sequence, wheremany genetic sequences are incorporated into a single sequence. Aconsensus sequence may thus minimize the genetic distance betweenvaccine strains and viruses and so may elicit more cross-reactive immuneresponses than an immunogen derived from any single influenza virus.

The consensus sequence approach is limited because the consensussequence is dependent on the input sequences, which are usually heavilybiased databases (e.g., temporal and spatial collection biases) based onhow sequences are reported to a database, such as the National Centerfor Biotechnology Information (NCBI). The sequences that are mostreported to NCBI are not necessarily representative of circulatingstrains. Consequently, the synthetic consensus sequence does notnecessarily represent currently circulating diversity. Moreover, since aconsensus sequence is 100% synthetic, it might not be functional orconformationally “correct”.

In contrast, the mosaic protein sequences disclosed herein weregenerated using an objective scoring mechanism that optimizes formaximum T cell epitope coverage of the known diversity of wild-typeinfluenza. Consequently, the synthetic protein that is generated is lesssubject to the inherent biases in the data. The utility of the mosaicantigen approach, and its superiority to a consensus sequence approach,was demonstrated in vivo for a mosaic H5 HA antigen (H5M). The H5Mvaccine can elicit protection against H5N1 HPAI clades 0, clade 1 andclade 2. Even recent consensus approaches that have tried to control forthe most diversity of input sequence have failed to simultaneouslyelicit immune responses against all of these clades (see, e.g., Hesselet al., 2011).

Exemplary Compositions and Methods of the Invention

The present invention relates to compositions and methods which employrecombinant nucleic acid sequence or vectors, or isolated protein, e.g.,HA or NA having at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% aminoacid sequence identity to one of SEQ ID NOs:1-14, e.g., a recombinantvirus or recombinant cell which expresses one or more of the recombinantgene products, or extracts of those cells, or inactivated recombinantvirus, e.g., inactivated via chemical or heat treatment, which expressesone or more of the protein(s). In one embodiment, the recombinant virusor isolated protein may be obtained from a recombinant bacterial cell,avian cell or mammalian cell.

The compositions and methods are useful for preventing, inhibiting ortreating influenza virus infection in animals including avians andmammals. The compositions of the invention, for example, a single dosethereof, are broad spectrum immunotherapeutics and provide forprophylactic and/or therapeutic activity against a variety of influenzavirus isolates. In one embodiment, the method includes administering acomposition of the invention to a mammal having or suspected of havingan influenza virus infection. In one embodiment, a composition comprisesan effective amount of recombinant isolated protein e.g., a proteinhaving at least 80%, 85%, 90%, 92%, 95%, 97%, 98%, or 99% amino acidsequence identity to one of SEQ ID NOs:1-14, or an immunogenic portionthereof, or a recombinant virus or cell, such as an attenuated oravirulent virus, which expresses one or more recombinant gene productsone of which is a mosaic protein of the invention, or soluble extractsof those cells. In one embodiment, the composition or method employs arecombinant vector that express a protein having at least 80%, 85%, 90%,92%, 95%, 97%, 98%, or 99% amino acid sequence identity to SEQ ID NO:1.

In one embodiment, the composition further comprises a pharmaceuticallyacceptable carrier. In one embodiment, the composition is administeredorally, for instance, in a formulation suitable to deliver protein(s).In another embodiment, the composition is administered through variousother acceptable delivery routes, for example, through parenteralinjection, intranasally, or via an intra-muscular injection. In oneembodiment, the composition is administered to the animal one or moretimes, at times including but not limited to 1 to 7 days, 1 to 3 weeksor about 1, 2, 3, 4 or more, e.g., up to 6, months, before the mammal oravian is exposed to influenza virus. In one embodiment, the compositionis administered to the mammal or avian one or more times after exposureto the virus, e.g., at 1 hour, 6 hours, 12 hours, 1 day, 2 days, 4 daysor more, e.g., up to about 2 weeks, after exposure.

In one embodiment, the invention provides a recombinant nucleic acidmolecule having a nucleotide sequence encoding an immunogenic influenzavirus HA polypeptide having one of SEQ ID NOs:1-11, a sequence with atleast 99% amino acid sequence identity thereto, or a portion thereof,which provides cross-clade reactivity. In one embodiment, the inventionprovides a recombinant nucleic acid molecule having a nucleotidesequence encoding an immunogenic H5 HA polypeptide having SEQ ID NO:1,or a sequence with at least 95% amino acid sequence identity thereto oran immunogenic portion thereof, wherein the HA has Ile at position 87,Thr at position 172, Val at position 226 or Thr at position 279, or acombination thereof. Further provided is a recombinant nucleic acidmolecule having a nucleotide sequence encoding an immunogenic H1 HApolypeptide having SEQ ID NO:2, 3, 4, or 5, a sequence with at least 95%amino acid sequence identity thereto or an immunogenic portion thereof,wherein the HA i) has Arg at position 206, Leu at position 432, or Valat position 434, or a combination hereof; ii) has Ile at position 125and Val at position 564; or iii) has Lys at position 62, Ile at position64, Gln at position 68, Asn at position 71, Ser at position 73, Val atposition 74, Leu at position 86, Ile at position 88, Ser at position 89,Lys at position 90, Glu at position 91, Lys at position 99, Pro atposition 100, Asn at position 101, Pro at position 102, Glu at position103, His at position 111, or Ala at position 113, or a combinationthereof. In addition, the invention provides a recombinant nucleic acidmolecule having a nucleotide sequence encoding an immunogenic H2 HApolypeptide having SEQ ID NO:6, a sequence with at least 95% amino acidsequence identity thereto or an immunogenic portion thereof, wherein theHA has Ala at position 24, Lys at position 45, Ser at position 87, Thrat position 258, Asn at position 260, or Leu at position 261, or acombination thereof. The invention also provides a recombinant nucleicacid molecule having a nucleotide sequence encoding an immunogenic H7 HApolypeptide having SEQ ID NO:8, a sequence with at least 95% amino acidsequence identity thereto or an immunogenic portion thereof, wherein theHA has Ser at position 91, Ser at position 92, Arg at position 122, Glyat position 127, Glu at position 195, Val at position 197, or Ser atposition 198, or a combination thereof, a recombinant nucleic acidmolecule having a nucleotide sequence encoding an immunogenic H9 HApolypeptide having SEQ ID NO:9, a sequence with at least 95% amino acidsequence identity thereto or an immunogenic portion thereof, wherein theHA has Gln at position 180, Glu at position 215, or Tyr at position 240,or a combination thereof, a recombinant nucleic acid molecule having anucleotide sequence encoding an influenza B HA polypeptide having SEQ IDNO:11, a sequence with at least 95% amino acid sequence identity theretoor an immunogenic portion thereof, wherein the HA has Met at position86, Val at position 88, Thr at position 90, Thr at position 91, Lys atposition 95, Ala at position 96, or Val at position 161, or acombination thereof, or a recombinant nucleic acid molecule having anucleotide sequence encoding a H3 HA polypeptide having SEQ ID NO:7,and/or a recombinant nucleic acid molecule having a nucleotide sequenceencoding a H10 HA polypeptide having having SEQ ID NO:10.

In one embodiment, the invention provides a recombinant nucleic acidmolecule having a nucleotide sequence encoding an immunogenic influenzavirus NA polypeptide having one of SEQ ID NOs:12-14, a sequence with atleast 95% amino acid sequence identity to SEQ ID NO:12 having Ala atposition 35, Ser at position 42, Asn at position 44, His at position 45,Thr at position 46, Gly at position 47, Ile at position 48, Arg atposition 52, Ser at position 59, His at position 64, Asn at position 70,Val at position 74, Val at position 75, Ala at position 76, Gly atposition 77, Asp at position 79, Lys at position 80, Thr at position 81,Ile at position 99, or Ser at position 105, or a combination thereof; asequence with at least 99% amino acid sequence identity to SEQ ID NO:13having Lys at position 199, Asn at position 221, or Gln at position 433,or a combination thereof; or a sequence with at least 99% amino acidsequence identity to SEQ ID NO:14 having Ile at position 353; or animmunogenic portion thereof.

The recombinant nucleic acid molecule may be in the form of anexpression vector, such as a recombinant virus, linked to the nucleotidesequence of the invention, e.g., forming a promoter operable in avian ormammalian cells, A recombinant poxvirus may include a nucleotidesequence encoding an immunogenic polypeptide having one of SEQ IDNOs.1-14, a sequence with at least 95% amino acid sequence identitythereto or a portion thereof that provides cross-clade reactivity.

The invention provides an isolated immunogenic influenza virus HApolypeptide having one of SEQ ID NOs:1-11, a sequence with at least 95%amino acid sequence identity thereto or a portion thereof, whichprovides cross-clade reactivity, e.g., the HA polypeptide has at leastor has greater than 99% amino acid sequence identity to one of SEQ IDNOs:1-11. In one embodiment, the polypeptide having SEQ ID NO:1, thesequence with at least 95% amino acid sequence identity thereto or theportion thereof, has Ile at position 87, Thr at position 172, Val atposition 226 or Thr at position 279, or a combination thereof. In oneembodiment, the polypeptide having SEQ ID NO:2, 3, 4, or 5, the sequencewith at least 95% amino acid sequence identity thereto or the portionthereof, i) has Arg at position 206, Leu at position 432, or Val atposition 434, or a combination hereof; ii) has Ile at position 125 andVal at position 564: or iii) has Lys at position 62, Ile at position 64,Gln at position 68, Asn at position 71, Ser at position 73, Val atposition 74, Leu at position 86, Ile at position 88, Ser at position 89,Lys at position 90, Glu at position 91, Lys at position 99, Pro atposition 100, Asn at position 101, Pro at position 102, Glu at position103, His at position 111, or Ala at position 113, or a combinationthereof. In one embodiment, the polypeptide having SEQ ID NO:6, asequence with at least 95% amino acid sequence identity thereto or animmunogenic portion thereof has Ala at position 24, Lys at position 45,Ser at position 86, Thr at position 258, Asn at position 260, or Leu atposition 261, or a combination thereof. In one embodiment, thepolypeptide having SEQ ID NO:8, the sequence with at least 95% aminoacid sequence identity thereto or the portion thereof, has Ser atposition 91, Ser at position 92, Arg at position 122, Gly at position127, Glu at position 195, Val at position 197, or Ser at position 198,or a combination thereof. In another embodiment, the polypeptide havingSEQ ID NO:9, the sequence with at least 95% amino acid sequence identitythereto or the portion thereof has Gln at position 180, Glu at position215, or Tyr at position 240, or a combination thereof. In yet anotherembodiment, the polypeptide having SEQ ID NO:11, the sequence with atleast 95% amino acid sequence identity thereto or the portion thereofhas Met at position 86, Val at position 88, Thr at position 90, Thr atposition 91, Lys at position 95, Ala at position 96, or Val at position161, or a combination thereof. The invention also provides an isolatedimmunogenic influenza virus NA polypeptide having one of SEQ IDNOs:12-14, a sequence with at least 95% amino acid sequence identity toSEQ ID NO: 12 or at least 99% amino acid sequence identity to one of SEQID NOs. 13-14, thereto or an immunogenic portion thereof. For example,the sequence with at least 95% amino acid sequence identity to SEQ IDNO:12 has Ala at position 35, Ser at position 42, Asn at position 44,His at position 45, Thr at position 46, Gly at position 47, Ile atposition 48, Arg at position 52, Ser at position 59, His a position 64,Asn at position 70, Val at position 74, Val at position 75, Ala atposition 76, Gly at position 77, Asp at position 79, Lys at position 80,Thr at position 81, Ile at position 99, or Ser at position 105, or acombination thereof; the sequence with at least 99% amino acid sequencesidentity to SEQ ID NO:13 has Lys at position 199, Asn at position 221,or Gln at position 433, or a combination thereof; or the sequence withat least 99% amino acid sequence identity to SEQ ID NO:14 has Ile atposition 353; or an immunogenic portion thereof.

The recombinant nucleic acid, e.g., a recombinant virus or recombinantpolypeptide may be employed as vaccine. In one embodiment, the inventionprovides a vaccine comprising a recombinant virus, the genome of whichcomprises at least one expression cassette having a promoter operablylinked to a heterologous open reading frame comprising a nucleotidesequence for an influenza virus polypeptide having one of SEQ ID NOs:1-14, a polypeptide with at least 95% amino acid sequence thereto or animmunogenic portion thereof, or a combination thereof, which providescross-clade reactivity. In one embodiment, the vaccine further comprisesan adjuvant. In one embodiment, the vaccine further comprises adifferent virus. In one embodiment, the vaccine further comprises apharmaceutically acceptable carrier, e.g., wherein the carrier issuitable for intranasal or intramuscular administration. In oneembodiment, the vaccine is in freeze-dried form. In one embodiment, thevaccine is adapted for mucosal, intramuscular or intradermal delivery.

Also provided is a method to prevent, inhibit or treat influenza virusinfection comprising administering to an avian or a mammal an effectiveamount of a composition comprising the recombinant nucleic acidmolecule, the recombinant virus, or the recombinant polypeptide of theinvention. Also provided is a recombinant method to immunize an animalagainst influenza infection, comprising: administering to an animal oran egg thereof, a composition comprising an amount of at least onerecombinant virus comprising a recombinant nucleic acid molecule of theinvention effective to induce an adaptive immune response to influenzavirus. In one embodiment, the animal is an avian or a mammal. In oneembodiment, the composition is intradermally administered. In oneembodiment, the composition is intramuscularly, or mucosally,administered. In one embodiment, the effective amount is administered inmore than one dose. In one embodiment, the composition further comprisesan adjuvant. In one embodiment, the composition is parenterallyadministered. In one embodiment, the composition is administeredintranasally or is administered orally.

In one embodiment, the invention provides a method to prevent influenzavirus infection of an animal. The method includes administering to amammal an effective amount of a live recombinant virus, the genome ofwhich comprises at least one expression cassette having a promoteroperably linked to a heterologous open reading frame comprising anucleotide sequence for a mosaic antigen of an influenza virus proteinthat elicits neutralizing antibodies and/or a cellular immune response.In one embodiment, the method includes administering to the mucosa of ananimal, e.g., orally administering, an effective amount of one or morelive recombinant poxviruses, the genome of at least one of whichcomprises at least one expression cassette having a promoter operablylinked to a heterologous open reading frame comprising a nucleotidesequence for a mosaic antigen that elicits neutralizing antibodiesand/or a cellular immune response. For example, the effective amount maybe from 1×10⁴ to 1×10⁸ PFU or TCID₅₀, e.g., from 1×10⁶ to 1×10⁷ PFU orTCID₅₀, which may be administered as a single dose or in two or moredoses, or each dose may include from 1×10⁴ to 1×10⁸ PFU or TCID₅₀, e.g.,from 1×10⁶ to 1×10⁷ PFU or TCID₅₀. For instance, each dose may have thesame number of PFU, or the booster dose(s) may have higher or loweramounts relative to the initial dose. The initial booster may beadministered from 2 to 8 weeks after the priming dose, for instance 3 to4 weeks after the priming dose. The priming dose and/or booster dose(s)may include an adjuvant. In one embodiment, mucosal delivery of therecombinant virus and adjuvant is employed.

Also provided is a method to immunize an avian or an egg thereof againstinfluenza virus. The method includes administering to the avian or anegg thereof an effective amount of isolated mosaic influenza virusprotein or a live recombinant virus, the genome of which comprises atleast one expression cassette having a promoter operably linked to aheterologous open reading frame comprising a nucleotide sequence for amosaic influenza virus protein that elicits neutralizing antibodiesand/or a cellular immune response. The immunized avian may be one of apopulation of avians, e.g., a flock of chickens, where at least one ofthe population has symptoms of infection or anti-influenza virusantibodies. In one embodiment, the antigen is a mosaic HA polypeptide.

In one embodiment, the method includes administering to the mucosa of anavian or mammal, e.g., orally or nasally administering, an effectiveamount of one or more recombinant viruses, the genome of at least one ofwhich comprises at least one expression cassette having a promoteroperably linked to a heterologous open reading frame comprising anucleotide sequence for a mosaic influenza virus antigen that elicitsneutralizing antibodies and/or a cellular immune response. For example,the effective amount may be from 1×10⁴ to 1×10⁸ PFU or TCID₅₀, e.g.,from 1×10⁶ to 1×10⁷ PFU or TCID₅₀, which may be administered as a singledose or in two or more doses, or each dose may include from 1×10⁴ to1×10⁸ PFU or TCID₅₀, e.g., from 1×10⁶ to 1×10⁷ PFU or TCID₅₀. Forinstance, each dose may have the same number of PFU, or the boosterdose(s) may have higher or lower amounts relative to the initial dose.The initial booster may be administered from 2 to 8 weeks after thepriming dose, for instance 3 to 4 weeks after the priming dose. Thepriming dose and/or booster dose(s) may include an adjuvant. In oneembodiment, mucosal delivery of the recombinant virus and adjuvant isemployed, e.g., a recombinant virus encoding influenza HA and anadjuvant, which adjuvant may be delivered via a recombinant virus.

In one embodiment, the invention provides a mosaic H5 polypeptidesequence with characteristic residues at positions 87, 172, 226 or 279,or a combination thereof, of HA (numbering of positions is that in aprotein having SEQ ID NO:1; in a HA sequence a signal peptide may befrom 15 to 20 residues in length, and the signal peptide in SEQ ID NO:1is 17 residues in length). For example, SEQ ID NO:1 has a 17 amino acidsignal peptide), e.g., the residue at position 87 of HA is notthreonine, the residue at position 172 is not alanine, the residue atposition 226 is not alanine, and/or the residue at position 279 is notalanine, or a combination thereof. In one embodiment, the isolated H5Mof the invention has a residue at position 87 with an aliphatic sidechain, e.g., Ile, a residue at position 172 with a hydroxyl side chain,e.g., Thr, a residue at position 226 with an aliphatic side chain, e.g.,Val, and/or a residue at position 279 with a hydroxyl side chain, e.g.,Thr. For example, a group of amino acids having aliphatic side chains isglycine, alanine, valine, leucine, and isoleucine; a group of aminoacids having aliphatic-hydroxyl side chains is serine and threonine; agroup of amino acids having amide-containing side chains is asparagineand glutamine; a group of amino acids having aromatic side chains isphenylalanine, tyrosine and tryptophan; a group of amino acids havingbasic side chains is lysine, arginine and histidine; and a group ofamino acids having sulfur-containing side chain is cysteine andmethionine. In one embodiment, conservative amino acid substitutiongroups are: threonine-valine-leucine-isoleucine-alanine;phenylalanine-tyrosine; lysine-arginine; alanine-valine;glutamic-aspartic; and asparagine-glutamine.

In one embodiment, the recombinant nucleic acid molecule or virus of theinvention encodes one or more influenza viral proteins (polypeptides)having at least 95%, e.g., 96%, 97%, 98% or 99%, amino acid sequenceidentity to one of SEQ ID NOs:1-14, so long as the polypeptide isimmunogenic. An amino acid sequence having at least 95%, e.g., 96%, 97%,98% or 99%, amino acid sequence identity includes sequences withdeletions or insertions, and/or substitutions, e.g., conservativesubstitutions, so long as the polypeptide is immunogenic. In oneembodiment, the one or more residues which are not identical may benonconservative substitutions. In one embodiment, the polypeptide hasone or more, for instance, 2, 5, 10, 15, 20 or more, amino acidsubstitutions, e.g., conservative substitutions of up to 5% of theresidues of the full-length, mature form of a polypeptide having SEQ IDNOs:1-14. In one embodiment, the isolated recombinant nucleic acidmolecule includes a nucleic acid sequence for the mosaic antigen thathas codon optimized sequences, a reduced number of RNA secondarystructures, a reduced number of RNA destabilization sequences, and/or areduced number of or no transcription terminator sequences, relative toan unmodified nucleotide sequence. The isolated recombinant nucleic acidmolecule or virus, or isolated mosaic polypeptide, of the invention maybe employed alone or with one or more other immunogenic agents, such asother virus in a vaccine, to raise virus-specific antisera, in genetherapy, and/or in diagnostics.

The isolated recombinant nucleic acid molecule of the invention may beemployed in a vector to express influenza proteins, e.g., forrecombinant protein vaccine production or to raise antisera, as anucleic acid vaccine, for use in diagnostics or, for vRNA production, toprepare chimeric genes, e.g., with other viral genes including otherinfluenza virus genes, and/or to prepare recombinant virus. Thus, theinvention also provides isolated viral polypeptides, recombinant virus,and host cells contacted (e.g., infected or transfected) with thenucleic acid molecule(s) and/or recombinant virus of the invention, aswell as isolated virus-specific antibodies, for instance, obtained frommammals infected with the virus or immunized with an isolated viralpolypeptide or polynucleotide encoding one or more viral polypeptides.

The invention also provides a method to induce an immune response in amammal, e.g., to immunize a mammal, or an avian against one moreinfluenza virus isolates. An immunological response to a composition orvaccine is the development in the host organism of a cellular and/orantibody-mediated immune response to a viral polypeptide, e.g., anadministered viral preparation, polypeptide or one encoded by anadministered nucleic acid molecule, which can prevent or inhibitinfection to closely structurally related viruses as well as moredistantly related viruses. Usually, such a response consists of thesubject producing antibodies, B cells, helper T cells, suppressor Tcells, and/or cytotoxic T cells directed specifically to an antigen orantigens included in the composition or vaccine of interest. The methodincludes administering to the host organism, e.g., a mammal, aneffective amount of the recombinant nucleic acid molecule, protein orvirus of the invention, e.g., an attenuated live virus, optionally incombination with an adjuvant and/or a carrier, e.g., in an amounteffective to prevent or ameliorate infection of an animal, such as amammal, by a plurality of different influenza viruses, e.g., fromdifferent clades and/or subtypes. In one embodiment, the virus isadministered intramuscularly while in another embodiment, the virus isadministered intranasally. In some dosing protocols, all doses may beadministered intramuscularly or intranasally, while in others acombination of intramuscular and intranasal administration is employed.The vaccine may further contain other recombinant viruses, otherantigens, additional biological agents or microbial components.

In one embodiment, a composition of the invention comprises one or moreisolated proteins, or recombinant virus or cells expressing one or moreproteins, including a protein of the invention, in an amount effectiveto elicit an anti-influenza virus response. For instance, recombinantprotein may be isolated from a suitable expression system, such asbacteria, insect cells or yeast, e.g., E. coli, L. lactis, Pichia or S.cerevisiae or other bacterial, insect or yeast expression systems, ormammalian expression systems such as T-REx™ (Invitrogen). For example,to prepare isolated recombinant proteins, any suitable host cell may beemployed, e.g., E. coli or yeast, or infected host cells, to expressthose proteins. Those cellular expression systems may also be employedas delivery systems, e.g., where the protein is one expressed on thecell surface or in a secreted form. A suitable cellular delivery systemmay be one for oral delivery. A recombinant protein useful in thecompositions and methods of the invention may be expressed on thesurface of a prokaryotic or eukaryotic cell, or may be secreted by thatcell, and may be expressed as a fusion or may be linked to a moleculethat alters solubility (e.g., prevents aggregation) or half-life, e.g.,a PEGylated molecule, of the resulting chimeric molecule. In oneembodiment, the composition of the invention may comprise a recombinantcell expressing one or more recombinant proteins, e.g., on the cellsurface or as a secreted protein.

Optimized Sequences

Also provided is an isolated nucleic acid molecule (polynucleotide)comprising a nucleic acid sequence which is optimized for expression inat least one selected host. Optimized sequences include sequences whichare codon optimized, i.e., codons which are employed more frequently inone organism relative to another organism, e.g., a distantly relatedorganism, or balance the usage of codons so that the most frequentlyused codon is not used to exhaustion. Other modifications can includeaddition or modification of Kozak sequences and/or introns, and/or toremove undesirable sequences, for instance, potential transcriptionfactor binding sites.

In one embodiment, the polynucleotide includes a nucleic acid sequenceencoding a mosaic antigen of the invention, which nucleic acid sequenceis optimized for expression in a mammalian host cell. In one embodiment,an optimized polynucleotide no longer hybridizes to a correspondingnon-optimized (wild-type) sequence, e.g., does not hybridize to thenon-optimized sequence under medium or high stringency conditions. Theterm “stringency” is used in reference to the conditions of temperature,ionic strength, and the presence of other compounds, under which nucleicacid hybridizations are conducted. With “high stringency” conditions,nucleic acid base pairing will occur only between nucleic acid fragmentsthat have a high frequency of complementary base sequences. Thus,conditions of “medium” or “low” stringency are often required when it isdesired that nucleic acids that are not completely complementary to oneanother be hybridized or annealed together. The art knows well thatnumerous equivalent conditions can be employed to comprise medium or lowstringency conditions.

Exemplary “high stringency conditions” when used in reference to nucleicacid hybridization comprise conditions equivalent to binding orhybridization at 42° C. in a solution consisting of 5×SSPE (43.8 g/lNaCl, 6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4 withNaOH), 0.5% SDS, 5×Denhardt's reagent and 100 μg/ml denatured salmonsperm DNA followed by washing in a solution comprising 0.1×SSPE, 1.0%SDS at 42° C. when a probe of about 500 nucleotides in length isemployed. Exemplary “medium stringency conditions” when used inreference to nucleic acid hybridization comprise conditions equivalentto binding or hybridization at 42° C. in a solution consisting of 5×SSPE(43.8 g/l NaCl, 6.9 g/l NaH₂PO₄H₂O and 1.85 g/l EDTA, pH adjusted to 7.4with NaOH), 0.5% SDS, 5×Denhardt's reagent and 100 μg/ml denaturedsalmon sperm DNA followed by washing in a solution comprising 1.0×SSPE,1.0% SDS at 42° C. when a probe of about 500 nucleotides in length isemployed.

In another embodiment, the polynucleotide has less than 90%, e.g., lessthan 80%, nucleic acid sequence identity to a correspondingnon-optimized (wild-type) sequence. Constructs, e.g., expressioncassettes, and vectors comprising the isolated nucleic acid molecule,e.g., with optimized nucleic acid sequence, as well as kits comprisingthe isolated nucleic acid molecule, construct or vector are alsoprovided.

A nucleic acid molecule comprising a nucleic acid sequence encoding amosaic antigen of the invention is optionally optimized for expressionin a particular host cell and also optionally operably linked totranscription regulatory sequences, e.g., one or more enhancers, apromoter, a transcription termination sequence or a combination thereof,to form an expression cassette.

In one embodiment, a nucleic acid sequence encoding a mosaic antigen ofthe invention is optimized by replacing codons, e.g., at least 25% ofthe codons, in a wild type sequence with codons which are preferentiallyemployed in a particular (selected) cell. Preferred codons have arelatively high codon usage frequency in a selected cell, and theirintroduction results in the introduction of relatively few undesirablestructural attributes. Thus, the optimized nucleic acid product may havean improved level of expression due to improved codon usage frequency,and a reduced number of undesirable transcription regulatory sequences.

An isolated and optimized nucleic acid molecule may have a codoncomposition that differs from that of the corresponding wild-typenucleic acid sequence at more than 30%, 35%, 40% or more than 45%, e.g.,50%, 55%, 60% or more of the codons. Exemplary codons for use in theinvention are those which are employed more frequently than at least oneother codon for the same amino acid in a particular organism and, in oneembodiment, are also not low-usage codons in that organism and are notlow-usage codons in the organism used to clone or screen for theexpression of the nucleic acid molecule. Moreover, codons for certainamino acids (i.e., those amino acids that have three or more codons),may include two or more codons that are employed more frequently thanthe other (non-preferred) codon(s). The presence of codons in thenucleic acid molecule that are employed more frequently in one organismthan in another organism results in a nucleic acid molecule which, whenintroduced into the cells of the organism that employs those codons morefrequently, is expressed in those cells at a level that is greater thanthe expression of the wild type or parent nucleic acid sequence in thosecells.

In one embodiment of the invention, the codons that are different arethose employed more frequently in a mammal. Codons for differentorganisms are known to the art, e.g., see www.kazusa.or.jp./codon/. Aparticular type of mammal, e.g., a human, may have a different set ofmore frequently employed codons than another type of mammal. In oneembodiment of the invention, at least a majority of the codons arecodons employed in mammals (e.g., humans). For example, codons employedmore frequently in humans include, but are not limited to, CGC (Arg),CTG (Leu), TCT (Ser), AGC (Ser), ACC (Thr), CCA (Pro), CCT (Pro), GCC(Ala), GGC (Gly), GTG (Val), ATC (Ile), ATT (Ile), MG (Lys), AAC (Asn),CAG (Gln), CAC (His), GAG (Glu), GAC (Asp), TAC (Tyr), TGC (Cys) and TTC(Phe). Thus, in one embodiment, nucleic acid molecules of the inventionhave a codon composition where at least a majority of codons arefrequently employed codons in humans, e.g., CGC, CTG, TCT, AGC, ACC,CCA, CCT, GCC, GGC, GTG, ATC, ATT, AAG, AAC, CAG, CAC, GAG, GAC, TAC,TGC, TTC, or any combination thereof. For example, the nucleic acidmolecule of the invention may CTG or TTG leucine-encoding codons, GTG orGTC valine-encoding codons, GGC or GGT glycine-encoding codons, ATC orATT isoleucine-encoding codons, CCA or CCT proline-encoding codons, CGCor CGT arginine-encoding codons, AGC or TCT serine-encoding codons, ACCor ACT threonine-encoding codon, GCC or GCT alanine-encoding codons, orany combination thereof. See FIG. 13 for codon usage tables for fourdifferent organisms.

Pharmaceutical Formulations

The compositions of this invention may be formulated with conventionalcarriers and excipients, which will be selected in accord with ordinarypractice. Aqueous formulations are prepared in sterile form, and whenintended for delivery by other than oral administration, will generallybe isotonic. All formulations will optionally contain excipients such asthose set forth in the Handbook of Pharmaceutical Excipients (1986).Excipients include ascorbic acid and other antioxidants, chelatingagents such as EDTA, carbohydrates such as dextrin,hydroxyalkylcellulose, hydroxyalkylmethylcellulose, stearic acid and thelike. The pH of the formulations ranges from about 3 to about 11, but isordinarily about 7 to 10 or about 8 to 9, e.g., for poxviruses.

While it is possible for the active ingredients to be administered alonethey may be present as pharmaceutical formulations. The formulations,both for veterinary and for human use, of the invention comprise atleast one active ingredient, as above defined, together with one or moreacceptable carriers therefor and optionally other therapeuticingredients. The carrier(s) must be “acceptable” in the sense of beingcompatible with the other ingredients of the formulation andphysiologically innocuous to the recipient thereof.

The formulations include those suitable for the foregoing administrationroutes. The formulations may conveniently be presented in unit dosageform and may be prepared by any of the methods well known in the art ofpharmacy. Techniques and formulations generally are found in Remington'sPharmaceutical Sciences (Mack Publishing Co., Easton, Pa.). Such methodsinclude the step of bringing into association the active ingredient withthe carrier which constitutes one or more accessory ingredients. Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredient with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

Formulations of the present invention suitable for oral administrationmay be presented as discrete units such as capsules, cachets or tabletseach containing a predetermined amount of the active ingredient; as apowder or granules; as a solution or a suspension in an aqueous ornon-aqueous liquid; or as an oil-in-water liquid emulsion or awater-in-oil liquid emulsion. The active ingredient may also beadministered as a bolus, electuary or paste.

Pharmaceutical formulations according to the present invention mayinclude one or more pharmaceutically acceptable carriers or excipientsand optionally other therapeutic agents. Pharmaceutical formulationscontaining the active ingredient may be in any form suitable for theintended method of administration. When used for oral use for example,tablets, troches, lozenges, aqueous or oil suspensions, dispersiblepowders or granules, emulsions, hard or soft capsules, syrups or elixirsmay be prepared. Compositions intended for oral use may be preparedaccording to any method known to the art for the manufacture ofpharmaceutical compositions and such compositions may contain one ormore agents including sweetening agents, flavoring agents, coloringagents and preserving agents, in order to provide a palatablepreparation.

Formulations for oral use may be also presented as hard gelatin capsuleswhere the active ingredient is mixed with an inert solid diluent, forexample calcium phosphate or kaolin, or as soft gelatin capsules whereinthe active ingredient is mixed with water or an oil medium, such aspeanut oil, liquid paraffin or olive oil.

Aqueous suspensions of the invention contain the active materials inadmixture with excipients suitable for the manufacture of aqueoussuspensions. Such excipients include a suspending agent, such as sodiumcarboxymethylcellulose, methylcellulose, hydroxypropyl methylcelluose,sodium alginate, polyvinylpyrrolidone, gum tragacanth and gum acacia,and dispersing or wetting agents such as a naturally occurringphosphatide (e.g., lecithin), a condensation product of an alkyleneoxide with a fatty acid (e.g., polyoxyethylene stearate), a condensationproduct of ethylene oxide with a long chain aliphatic alcohol (e.g.,heptadecaethyleneoxycetanol), a condensation product of ethylene oxidewith a partial ester derived from a fatty acid and a hexitol anhydride(e.g., polyoxyethylene sorbitan monooleate). The aqueous suspension mayalso contain one or more preservatives such as ethyl or n-propylp-hydroxy-benzoate, one or more coloring agents, one or more flavoringagents and one or more sweetening agents, such as sucrose or saccharin.

Oil suspensions may be formulated by suspending the active ingredient ina vegetable oil, such as arachis oil, olive oil, sesame oil or coconutoil, or in a mineral oil such as liquid paraffin. The oral suspensionsmay contain a thickening agent, such as beeswax, hard paraffin or cetylalcohol. Sweetening agents, such as those set forth above, and flavoringagents may be added to provide a palatable oral preparation. Thesecompositions may be preserved by the addition of an antioxidant such asascorbic acid.

The amount of active ingredient that may be combined with the carriermaterial to produce a single dosage form will vary depending upon thehost treated and the particular mode of administration. For example, atime-release formulation intended for oral administration to humans maycontain approximately 1 to 1000 mg of active material compounded with anappropriate and convenient amount of carrier material which may varyfrom about 5 to about 95% of the total compositions (weight:weight). Thepharmaceutical composition can be prepared to provide easily measurableamounts for administration. For example, an aqueous solution intendedfor intravenous infusion may contain from about 3 to 500 μg of theactive ingredient per milliliter of solution in order that infusion of asuitable volume at a rate of about 30 mL/hr can occur.

Formulations suitable for intrapulmonary or nasal administration mayhave a particle size for example in the range of 0.1 to 500 microns(including particle sizes in a range between 0.1 and 500 microns inincrements microns such as 0.5, 1, 30 microns, 35 microns, etc.), whichis administered by rapid inhalation through the nasal passage or byinhalation through the mouth so as to reach the alveolar sacs. Suitableformulations include aqueous or oily solutions of the active ingredient.Formulations suitable for aerosol or dry powder administration may beprepared according to conventional methods and may be delivered withother therapeutic agents such as compounds heretofore used in thetreatment or prophylaxis of a given condition.

Formulations suitable for parenteral administration include aqueous andnon-aqueous sterile injection solutions which may contain anti-oxidants,buffers, bacteriostats and solutes which render the formulation isotonicwith the blood of the intended recipient; and aqueous and non-aqueoussterile suspensions which may include suspending agents and thickeningagents.

The formulations may be presented in unit-dose or multi-dose containers,for example sealed ampoules and vials, and may be stored in afreeze-dried (lyophilized) condition requiring only the addition of thesterile liquid carrier, for example water for injection, immediatelyprior to use. Extemporaneous injection solutions and suspensions areprepared from sterile powders, granules and tablets of the kindpreviously described. Exemplary unit dosage formulations are thosecontaining a daily dose or unit daily sub-dose, as herein above recited,or an appropriate fraction thereof, of the active ingredient.

It should be understood that in addition to the ingredients particularlymentioned above the formulations of this invention may include otheragents conventional in the art having regard to the type of formulationin question, for example those suitable for oral administration mayinclude flavoring agents.

The invention further provides veterinary compositions comprising atleast one active ingredient as above defined together with a veterinarycarrier therefor.

Veterinary carriers are materials useful for the purpose ofadministering the composition and may be solid, liquid or gaseousmaterials which are otherwise inert or acceptable in the veterinary artand are compatible with the active ingredient. These veterinarycompositions may be administered orally, parenterally or by any otherdesired route.

Pharmaceutical Compositions

Pharmaceutical compositions of the present invention, suitable forinoculation, e.g., nasal, ocular, parenteral or oral administration,comprise one or more recombinant nucleic acid molecules, virus isolates,and/or isolated protein of the invention, optionally further comprisingsterile aqueous or non-aqueous solutions, suspensions, and emulsions.The compositions can further comprise auxiliary agents or excipients, asknown in the art. The composition of the invention is generallypresented in the form of individual doses (unit doses).

For example, for influenza virus vaccines, conventional vaccinesgenerally contain about 0.1 to 200 μg, e.g., 30 to 100 μg or 15 to about100 ug, of influenza virus HA from each of the strains entering intotheir composition. The vaccine forming the main constituent of thevaccine composition of the invention may comprise a single virusencoding an influenza virus mosaic antigen, or one or more virusesencoding antigens from a combination of subtypes or combination ofantigens, for example, at least two or three different influenza virusantigens, one of which is a mosaic antigen.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and/or emulsions, which may containauxiliary agents or excipients known in the art. Examples of non-aqueoussolvents are propylene glycol, polyethylene glycol, vegetable oils suchas olive oil, and injectable organic esters such as ethyl oleate.Carriers or occlusive dressings can be used to increase skinpermeability and enhance antigen absorption. Liquid dosage forms fororal administration may generally comprise a liposome solutioncontaining the liquid dosage form. Suitable forms for suspendingliposomes include emulsions, suspensions, solutions, syrups, and elixirscontaining inert diluents commonly used in the art, such as purifiedwater. Besides the inert diluents, such compositions can also includeadjuvants, wetting agents, emulsifying and suspending agents, orsweetening, flavoring, or perfuming agents.

As will be apparent to one skilled in the art, the optimal concentrationof the active agent in a composition of the invention will necessarilydepend upon the specific agent(s) used, the characteristics of the avianor mammal, the type and amount of adjuvant, if any, and/or the nature ofthe infection. These factors can be determined by those of skill in themedical and pharmaceutical arts in view of the present disclosure.

Specific dosages may be adjusted depending on conditions of disease, theage, body weight, ethnic background, general health conditions, sex,diet, lifestyle and/or current therapeutic regimen of the mammal, aswell as for intended dose intervals, administration routes, excretionrate, and combinations of drugs. Any of the dosage forms describedherein containing effective amounts are well within the bounds ofroutine experimentation and therefore, well within the scope of theinstant disclosure.

In addition to the recombinant virus, recombinant cells or isolatedprotein, or combinations thereof, the composition of the invention mayfurther comprise one or more suitable pharmaceutically acceptablecarriers. As used herein, the term “pharmaceutically acceptable carrier”refers to an acceptable vehicle for administering a composition tomammals comprising one or more non-toxic excipients which do not reactwith or reduce the effectiveness of the pharmacologically active agentscontained therein. The proportion and type of pharmaceuticallyacceptable carrier in the composition may vary, depending on the chosenroute of administration. Suitable pharmaceutically acceptable carriersfor the compositions of the present disclosure are described in thestandard pharmaceutical texts. See, e.g., “Remington's PharmaceuticalSciences”, 18^(th) Ed., Mack Publishing Company, Easton, Pa. (1990).Specific non-limiting examples of suitable pharmaceutically acceptablecarriers include water, saline, dextrose, glycerol, ethanol, or the likeand combinations thereof.

Optionally, the composition may further comprise minor amounts ofauxiliary substances such as agents that enhance the effectiveness ofthe preparation, stabilizers, preservatives, and the like.

In one embodiment, the composition may also comprise a bile acid or aderivative thereof, in particular in the form of a salt. These includederivatives of cholic acid and salts thereof, in particular sodium saltsof cholic acid or cholic acid derivatives. Examples of bile acids andderivatives thereof include cholic acid, deoxycholic acid,chenodeoxycholic acid, lithocholic acid, ursodeoxycholic acid,hyodeoxycholic acid and derivatives such as glyco-, tauro-,amidopropyl-1-propanesulfonic-, amidopropyl-2-hydroxy-1-propanesulfonicderivatives of the aforementioned bile acids, orN,N-bis(3Dgluconoamidopropyl)deoxycholamide. A particular example issodium deoxycholate (NaDOC).

Examples of suitable stabilizers include protease inhibitors, sugarssuch as sucrose and glycerol, encapsulating polymers, chelating agentssuch as ethylene-diaminetetracetic acid (EDTA), proteins andpolypeptides such as gelatin and polyglycine and combinations thereof.

Optionally, the composition may further comprise an adjuvant in additionto the recombinant virus, recombinant cells or isolated proteindescribed herein. Suitable adjuvants for inclusion in the compositionsof the present disclosure include those that are well known in the art,such as complete Freund's adjuvant (CFA) that is not used in humans,incomplete Freund's adjuvant (IFA), squalene, squalane, alum, andvarious oils, all of which are well known in the art, and are availablecommercially from several sources, such as Novartis (e.g., Novartis'MF59 adjuvant).

Depending on the route of administration, the compositions may take theform of a solution, suspension, emulsion, or the like. A composition ofthe invention can be administered intranasally or through enteraladministration, such as orally, or through subcutaneous injection,intra-muscular injection, intravenous injection, intraperitonealinjection, or intra-dermal injection to a mammal, e.g., humans, horses,other mammals, etc. Compositions may be formulated for a particularroute of delivery, e.g., formulated for oral delivery.

For parenteral administration, the composition of the invention may beadministered by intravenous, subcutaneous, intramuscular,intraperitoneal, or intradermal injection, and may further comprisepharmaceutically accepted carriers. For administration by injection, thecomposition may be in a solution in a sterile aqueous vehicle which mayalso contain other solutes such as buffers or preservatives as well assufficient quantities of pharmaceutically acceptable salts or of glucoseto make the solution isotonic.

The composition may be delivered to the respiratory system, for exampleto the nose, sinus cavities, sinus membranes or lungs, in any suitablemanner, such as by inhalation via the mouth or intranasally. Thecomposition may be dispensed as a powdered or liquid nasal spray,suspension, nose drops, a gel or ointment, through a tube or catheter,by syringe, by packtail, by pledget, or by submucosal infusion. Thecomposition may be conveniently delivered in the form of an aerosolspray using a pressurized pack or a nebulizer and a suitable propellant,e.g., without limitation, dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane or carbon dioxide. Inthe case of a pressurized aerosol, the dosage unit may be controlled byproviding a valve to deliver a metered amount. Capsules and cartridgesof, for example, gelatin for use in an inhaler or insufflator may beformulated containing a powder mix of the composition and a suitablepowder base such as lactose or starch. Examples of intranasalformulations and methods of administration can be found in PCTpublications WO 01/41782, WO 00/33813, and U.S. Pat. Nos. 6,180,603;6,313,093; and 5,624,898, all of which are incorporated herein byreference and for all purposes. A propellant for an aerosol formulationmay include compressed air, nitrogen, carbon dioxide, or a hydrocarbonbased low boiling solvent. The composition of the invention may beconveniently delivered in the form of an aerosol spray presentation froma nebulizer or the like. In some aspects, the active ingredients aresuitably micronized so as to permit inhalation of substantially all ofthe active ingredients into the lungs upon administration of the drypowder formulation, thus the active ingredients will have a particlesize of less than 100 microns, desirably less than 20 microns, such asin the range 1 to 10 microns or 0.2 to 0.4 microns. In one embodiment,the composition is packaged into a device that can deliver apredetermined, and generally effective, amount of the composition viainhalation, for example a nasal spray or inhaler.

The vaccines of the present disclosure may further comprise one or moresuitable pharmaceutically acceptable carriers. As used herein, the term“pharmaceutically acceptable carrier” refers to an acceptable vehiclefor administering a vaccine to mammals comprising one or more non-toxicexcipients which do not react with or reduce the effectiveness of thepharmacologically active agents contained therein. The proportion andtype of pharmaceutically acceptable carrier in the vaccine may vary,depending on the chosen route of administration. Suitablepharmaceutically acceptable carriers for the vaccines of the presentdisclosure are described in the standard pharmaceutical texts. See,e.g., “Remington's Pharmaceutical Sciences”, 18^(th) Ed., MackPublishing Company, Easton, Pa. (1990). Specific non-limiting examplesof suitable pharmaceutically acceptable carriers include saline (e.g.,PBS), dextrose, glycerol, or the like and combinations thereof.

In addition, if desired, the vaccine can further contain minor amountsof auxiliary substances such as agents that enhance the antiviraleffectiveness of the composition, stabilizers, preservatives, and thelike.

Depending on the route of administration, the vaccine may take the formof a solution, suspension, emulsion, or the like. A vaccine of thepresent disclosure can be administered orally, intranasally, or throughparenteral administration, such as through sub-cutaneous injection,intra-muscular injection, intravenous injection, intraperitonealinjection, or intra-dermal injection to a mammal, e.g., humans, horses,other mammals, etc. Typically, the vaccine is administered throughintramuscular or intradermal injection, or orally.

For parenteral administration, the vaccines of the present disclosuremay be administered by intravenous, subcutaneous, intramuscular,intraperitoneal, or intradermal injection, which optionally may furthercomprise pharmaceutically accepted carriers. For administration byinjection, the vaccine may be a solution in a sterile aqueous vehiclewhich may also contain other solutes such as buffers or preservatives aswell as sufficient quantities of pharmaceutically acceptable salts or ofglucose to make the solution isotonic.

The vaccine may be delivered locally to the respiratory system, forexample to the nose, sinus cavities, sinus membranes or lungs, in anysuitable manner, such as by inhalation via the mouth or intranasally.The vaccines can be dispensed as a powdered or liquid nasal spray,suspension, nose drops, a gel or ointment, through a tube or catheter,by syringe, by packtail, by pledget, or by submucosal infusion. Thevaccines may be conveniently delivered in the form of an aerosol sprayusing a pressurized pack or a nebulizer and a suitable propellant, e.g.,without limitation, dichlorodifluoromethane, trichlorofluoromethane,dichlorotetrafluoroethane or carbon dioxide. In the case of apressurized aerosol, the dosage unit may be controlled by providing avalve to deliver a metered amount. Capsules and cartridges of, forexample, gelatin for use in an inhaler or insufflator may be formulatedcontaining a powder mix of the vaccine and a suitable powder base suchas lactose or starch. Examples of intranasal formulations and methods ofadministration can be found in PCT publications WO 01/41782, WO00/33813, and U.S. Pat. Nos. 6,180,603; 6,313,093; and 5,624,898, all ofwhich are incorporated herein by reference and for all purposes. Apropellant for an aerosol formulation may include compressed air,nitrogen, carbon dioxide, or a hydrocarbon based low boiling solvent.The vaccines of the present disclosure can be conveniently delivered inthe form of an aerosol spray presentation from a nebulizer or the like.In some aspects, the active ingredients are suitably micronized so as topermit inhalation of substantially all of the active ingredients intothe lungs upon administration of the dry powder formulation, thus theactive ingredients will have a particle size of less than 100 microns,desirably less than 20 microns, and preferably in the range 1 to 10microns or 0.2 to 0.4 microns. In one embodiment, the vaccine ispackaged into a device that can deliver a predetermined, and generallyeffective, amount of the vaccine via inhalation, for example a nasalspray or inhaler.

The vaccines of the present disclosure are administeredprophylactically. For instance, administration of the vaccine may becommenced before or at the time of infection. In particular, thevaccines may be administered up to about 1 month or more, or moreparticularly up to about 4 months or more before the mammal is exposedto the microbe. Optionally, the vaccines may be administered as soon as1 week before infection, or more particularly 1 to 5 days beforeinfection.

The desired vaccine dose may be presented in a single dose or as divideddoses administered at appropriate intervals, for example as two, three,four or more sub-doses per day. Optionally, a dose of vaccine may beadministered on one day, followed by one or more booster doses spaced asdesired thereinafter. In one exemplary embodiment, an initialvaccination is given, followed by a boost of the same vaccineapproximately one week to 15 days later.

The dosage of a live virus vaccine for an animal such as a mammalianadult organism can be from about 10²-10¹⁵, e.g., 10³-10¹², plaqueforming units (PFU)/kg, or any range or value therein. For poxvirusesthat express influenza virus HA, the dosage of PFU or immunoreactive HAin each dose of replicated virus vaccine may be standardized to containa suitable amount, e.g., 30 to 100 μg, such as 15 to 100 ug, or anyrange or value therein, or the amount recommended by government agenciesor recognized professional organizations. If the poxvirus expresses adifferent influenza virus protein, that protein may be standardized. Forexample, the quantity of NA may also be standardized, however, thisglycoprotein may be labile during purification and storage.

The invention will be further described by the following non-limitingexamples.

Example I Materials and Methods Cells and Viruses

Chicken embryo fibroblasts (CEFs) and Mardin-Darby canine kidney (MDCK)cells were obtained from Charles River Laboratories, Inc. (Wilmington,Wash.) and the American Type Culture Collection (ATCC, Manassas, Va.),respectively. Cells were cultured in Dulbecco's Modified Eagle's Medium(DMEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics.CEFs were used for propagating MVA virus. Highly pathogenic avianinfluenza (H5N1) virus A/Vietnam/1203/04 was kindly provided by Dr.Yoshihiro Kawaoka (University of Wisconsin-Madison, Wis., USA). Highlypathogenic avian influenza (H5N1) viruses, A/Hongkong/483/97,A/Mongolia/Whooper swan/244/05 and A/Egypt/1/08 and seasonal influenzaviruses including A/Puerto Rico/8/34 (PR8, H1N1) and A/Aichi/2/1968(H₃N₂) were kindly provided by Dr. Stacey Schultz-Chemy and Dr. GhaziKayali (St. Jude children's research hospital, Memphis, Term.). Allviruses were propagated and titrated in MDCK cells with DMEM thatcontained 1% bovine serum albumin and 20 Mm HEPES. Viruses were storedat −80° C. until use. Viral titers were determined and expressed as 50%tissue culture infective dose (TCID₅₀). All experimental studies withHPAI H5N1 viruses were conducted in a BSL3+ facility in compliance withthe UW Madison Office of Biological Safety.

Plasmid and MVA Recombinant Vaccine Construction.

A total of 3,069 HA protein sequences from H5N1 viruses available inNational Center for Biotechnology Information (NCBI) database weredownloaded and screened to exclude incomplete and redundant sequences.The resulting 2,145 HA sequences were selected to generate one mosaicprotein sequence as previously described (Fischer et al., 2007). T cellepitopes were set to 12 amino acid length (12-mer) in an attempt tomatch the length of natural T helper cell epitopes (Goglak et al.,2000). The resulting mosaic H5N1 sequence (H5M) was back-translated andcodon optimized for mice. The optimized H5M sequence was thensynthesized commercially (GenScript USA Inc.) and cloned intoMVA-shuttle vector (Brewoo et al., 2013). Recombinant MVA expressing amosaic H5 (MVA-H5M) was generated in CEF cells as described elsewhere(Earl et al., 2001a; Earl et al., 2001b). MVA expressing wild type HAfrom avian influenza A/VN/1203/04 (MVA-HA) was constructed as describedin Brewor et al. (2013) and kindly provided by Inviragen, Inc.

Analysis of HA Expressed by H5M

The hemagglutin expressed by MVA-H5M was analyzed by western blotanalysis. CEF cells were infected with 1 multiplicity of infection (MOI)of 1 PFU/cell of MVA-H5M, MVA-HA and MVA-LUC constructs. Infected cellpellets were harvested 48 hours post-infection and lysed with Laemmlisample buffer (BioRad). Protein was fractionated via SDS PAGE andproteins were transferred onto nitrocellulose membrane for hemaggutinindetection by specific anti-HA antibody. 3,3′,5,5′-tetramethylbenzidine(TMB) was used to visualized HA protein in the membranes.

Functional analysis of H5M was done by hemagglutination assay (Killian(2008)). CEF cells were infected with 1 MOI of 1 PFU/cell of MVA-H5M,MVA-HA and MVA-LUC. After 48 hours post-infection, cells were harvestedand 2-fold dilutions with PBS were made in round bottom 96 well plates.Chicken red blood cells were added into each well and incubated for 30minutes. Lattice formations were observed in positive wells which isindicative of the ability of HA to agglutinate RBC.

Animal Studies

All mouse studies were conducted at University of Wisconsin-Madisonanimal facilities and were approved by the Inter-institutional AnimalCare and Use Committee (IACUC). Challenge experiments involving H5N1viruses were conducted at ABSL3+ facilities. Challenge studies forseasonal influenza, A/Puerto Rico/8/34 (PR8, H1N1), and A/Aichi/2/1968(H3N2) were conducted under the BSL2 conditions to facilitate animalmonitoring.

Vaccine Efficacies

Groups of 5 week-old BALB/c mice were vaccinated with 1×10⁷ plaqueforming unit (pfu) of either recombinant MVA-H5M, or MVA-expressingluciferase (MVA-LUC) via the intradermal (ID) route. Intradermalinoculations were done by injecting 50 μL of PBS-containing virus intofootpads. Four weeks after vaccination, blood samples were collected forserological analysis. At week five post vaccination, mice werechallenged by intranasal (IN) instillation under isoflurane anesthesiawith 100 LD₅₀ of A/Vietnam/1203/04 (1×10⁴ TCID₅₀), A/Hongkong/483/97(4×10³ TCID₅₀), A/Mongolia/Whooper swan/244/05 virus (1×10³ TCID₅₀) orA/Egypt/1/08 (3.56×10⁴ TCID₅₀) contained in 20 μL of PBS. Two mice fromeach group were euthanized at day five post-challenge and lung tissueswere collected for viral titrations and histopathology. For isolation ofvirus, lung tissues were minced in PBS using a mechanical homogenizer(MP Biochemicals, Solon, Ohio), and viral titers in homogenates werequantified by plaque assay on MDCK cells. The remaining lung tissue wasfixed in 10% formalin. The remaining animals in each group were observeddaily for 14 days, and survival and clinical parameters includingclinical score and body weight were recorded. Mice showing at least 20%body weight loss were humanely euthanized.

A second study evaluated the protective efficacies of the MVA-H5Mvaccine against seasonal influenza virus, PR8 (H1N1) or A/Aichi/2/1968(H₃N₂). Groups of 5 week-old BALB/c mice were vaccinated with MVA-H5M orMVA-LUC as above. Four weeks after vaccination, blood samples werecollected for serological analysis. At week five post vaccination, micewere challenged by intranasal (IN) instillation under isofluoraneanesthesia with 50 μL of PBS containing 100 LD₅₀ of PR8 (6.15×10³TCID₅₀) or A/Aichi/2/1968 (5×10⁶ TCID₅₀). Two mice from each group wereeuthanized at day three post-challenge and lung tissues were collectedas above. The remaining animals in each group were observed daily for 14days as described above.

Serology

Serum antibody titers were determined by microneutralization assay.Briefly, serum was incubated at 56° C. for 30 minutes to inactivatecomplement and then serially diluted two fold in microtiter plates. 200TCID₅₀ units of virus were added to each well and incubated at 37° C.for 1 hour. The virus-serum mixture was added to duplicate wells of MDCKcell in 96-well plates, incubated at 37° C. for 72 hours, then fixed andstained with 10% (W/V) crystal violet in 10% (V/V) formalin to determinethe TCID₅₀. The titer was determined as the serum dilution resulting inthe complete neutralization of the virus.

Histopathology and Immunohistochemistry

Lung samples for histological analysis were processed by thehistopathology laboratory at The School of Veterinary Medicine,(UW-Madison, Wis.) and stained with H&E. For immunohistochemistry,tissue sections were deparaffinized and rehydrated as previouslydescribed (Brown et al., 1992; Chamnanpood et al., 2011). Slides weretreated with antigen retrieval buffer followed up with 3% H₂O₂. Slideswere placed in blocking solution and incubated in goat-anti-HA (BEIresource #NR-2705) (1/300 dilution) avian influenza polyclonal antibodyfor 24 hours. Secondary HRP-conjugated anti-goat antibody at 1/5000diluted were added onto slides and incubated for 1 hour. Then, slideswere stained with 0.05% 3,3′-diaminobenzidine (DAB) substrate tovisualize the presence of avian influenza antigens.

T Cell Responses

At five months post-vaccination, two MVA-H5M vaccinated mice wereeuthanized and spleens were aseptically removed. Splenocytes fromindividual animals were suspended in RPMI-1640 medium supplemented with10% heat-inactivated fetal calf serum, 100 I.U./mL penicillin, 100 μg/mLstreptomycin and 0.14 mM β-mercaptoethanol. Red blood cells were lysedwith 1×BD Pharm Lyse™ buffer. Following washing with RPMI medium, cellswere resuspended in the same medium and 1×10⁶ splenocytes were surfacestained with anti-mouse CD4 FITC (RM4-5) and anti-mouse CD8a PerCP(53-6.7) mAbs. In order to study intracellular cytokine responses, 1×10⁶splenocytes were plated onto a 96-well flat-bottom plate and stimulatedwith diverse H5N1 HA peptide pools (5 μg/mL) in 200 μl total volume for16 hours. Bredfeldin A (BD GolgiPlug) was added at a final concentrationof 1 μg/ml for the last 5 hrs of incubation to block protein transport.Cells were stained intracellularly for IFN-γ APC (XMG1.2) and IL-2 PE(JES6-5H4) after surface staining for CD4 and CD8a. All antibodies werefrom BD Bioscience except where noted. The samples were acquired on BDFACSCalibur and analyzed with FlowJo v10.0.6 (Tree star). The cytokinebackground from medium-treated groups was subtracted from each sample.The frequency of cytokine-positive T cells was presented as thepercentage of gated CD4⁺ or CD8⁺ T cells.

Statistical Analysis

Student's T-tests were used to evaluate viral lung titers and antibodytiters between groups. Survival analyses were performed to assessvaccine effectiveness against challenge viruses. Probability values<0.05 were considered significant. GraphPad Prism 6 software (La jolla,CA) was used for all statistical analyses.

Results

The use of a “genetic algorithm” (e.g., “Mosaic Vaccine Tool Suite,”developed by Los Alamos National Labs for HIV work) to generate, select,and “recombine” (in silico) potential T and B cell epitopes (about 9-12amino acids in length) into “mosaic” proteins, can provide greatercoverage of global viral variants, and thus optimized immunogenicity,than any single wild-type protein. The mosaic sequence accounts for thecomplete or full-length sequence of the protein and/or regions ofinterest, as well as the full diversity of the ‘core’ sequencesprovided. The use of a mosaic sequence for an HIV-1 vaccine, whichrecombined potential T cell epitopes into Gag, Pol and Env proteins, hasbeen reported (Fischer et al., 2007). Mosaic HIV-1 vaccines expanded thebreadth and depth of cellular immune responses in rhesus monkeyscompared to consensus sequences (Barouch et al., 2010).

FIG. 2 is a schematic of an exemplary mosaic vaccine approach. Naturalsequences, e.g., from field isolates and not from viruses passed inculture, that represent the diversity found in current or currentlycirculating strains, and that represent a specific subtype are selected.Repeats of sequences are eliminated. Recombined sequence populations(about 500) are generated in silico and the coverage of a sequence fromeach population is compared to the natural sequence, e.g., as if it werethe representative sequence for the population. A representative mosaicsequence from each population is evaluated for its fitness.Representative sequences with rare T cell epitopes are generallyexcluded. To further evolve the sequences, parent mosaic sequences,e.g., pairs of random parental sequences, can be recombined in silico togenerate child sequences. The fitness of one or more random childsequences is/are determined and if the fitness has better coverage ofthe input sequences, the parental with the lowest score is replaced withthe higher scoring child sequence or, if the child score is the highestfor the population, it is the representative for that population. Thescoring of representative sequences in a population may be repeateduntil the fitness is no longer being improved, e.g., for a number ofcycles such as 10 cycles.

Construction of Pox-Based H5N1 Mosaic Hemagglutinin Vaccine

A mosaic vaccine that targets the hemagglutinin protein of influenzaH5N1 virus was constructed. The HA mosaic was generated using an inputof 2,145 HA sequences from H5N1 influenza viruses available in GenBank.To maximize T helper cell epitope coverage, the in silico algorithm wasset to an amino acid length of 12 mer (Gogolak et al., 2000). HAsequences from H5N1 strains (2,145 sequences) were used to generate amosaic sequence (FIG. 3):

MEKIVLLLAIVSLVKSDQICIGYHANNSTEQVDTIMEKNVTVTHAQDILEKTHNGKLCDLDGVKPLILRDCSVAGWLLGNPMCDEFINVPEWSYIVEKANPANDLCYPGNFNDYEELKHLLSRINHFEKIQIIPKSSWSDHEASSGVSSACPYQGRSSFFRNVVWLIKKNSTYPTIKRSYNNTNQEDLLVLWGIHHPNDAAEQTRLYQNPTTYISVGTSTLNQRLVPKIATRSKVNGQSGRMEFFWTILKPNDAINFESNGNFIAPEYAYKIVKKGDSTIMKSELEYGNCNTKCQTPMGAINSSMPFHNIHPLTIGECPKYVKSNRLVLATGLRNSPQRERRRKKRGLFGAIAGFIEGGWQGMVDGWYGYHHSNEQGSGYAADKESTQKAIDGVTNKVNSIIDKMNTQFEAVGREFNNLERRIENLNKKMEDGFLDVWTYNAELLVLMENERTLDFHDSNVKNLYDKVRLQLRDNAKELGNGCFEFYHKCDNECMESVRNGTYDYPQYSEEARLKREEISGVKLESIGTYQILSIYSTVASSLALAIMVAGLSLWMCSNGSLQCRICI (SEQ ID NO:1). The mosaic H5 (H5M) sequence was back-translated intoDNA and cloned into Modified Vaccinia Ankara (MVA) to generate MVA-H5M.That nucleotide sequence may be altered to improve expression, e.g., bycodon optimization for mammalian cells such as mice (the model fortesting), eliminating RNA secondary structure, eliminating RNAdestabilization sequences, removing transcription termination sequences(e.g., TTTTTNT), and/or adding a Kozak sequence to the 5′ end. Thepoxvirus encoding H5M was used to infect chicken embryo fibroblasts andsupernatants that were harvested 48 hours after infection were analyzedby Western blot (FIG. 5). Recombinant H5M was expressed as cleavable HAthat resembled a wild-type (wt) HA from avian influenza A/VN/1203/04.Interestingly, the level of protein expression from MVA-H5M infectedcell pellets was higher than the MVA expressing wild-type hemagglutininfrom ANN/1203/04 (MVA-HA) (Brewoo et al., 2013) (FIG. 5). Additionally,in vitro functional analysis of H5M resulted in hemaglutination of RBCat similar levels to wt HA (data not shown). These data thus demonstratethe successful generation of an MVA vector expressing a mosaic H5N1 HAgene.

Efficacy of MVA-H5M Vaccine Against Influenza Viruses

To determine whether antibodies to MVA-H5M had virus neutralizingactivity, mice were intradermally inoculated with MVA-H5M, MVA-LUC, orPBS; and antibody titers from immunized mice were measured againstA/VN/1203/04, A/MG/244/05, A/HK/483/97 and A/Egypt/1/08 challengeviruses. At four weeks post-immunization, MVA-H5M elicited significantneutralizing antibody (Ab) titers and protected against all four H5N1strains (FIGS. 5B & 13A-C&G). Geometric mean titers (GMT) of Ab againstA/VN/1203/04, A/MG/244/05 and A/HK/483/97 did not differ significantly.In contrast, GMT of Ab against A/Egypt/1/08 was significantly lower thanfor the other three H5N1 viruses. None of the MVA-LUC control injectedanimals survived challenge (FIGS. 13A-C and G). Notably, no virusreplication was detected in the lungs of any of the MVA-H5M vaccinatedmice challenged with any of the four H5N1 viruses (FIG. 13M).Furthermore, vaccination with MVA-H5M reduced lung pathology afterchallenge with avian influenza viruses (FIG. 16). MVA-H5M-vaccinatedmice showed no to mild lung lesions compared to the MVA-LUC-vaccinatedgroups. Lesions included thickening of alveolar wall, lung consolidationwith white blood cell infiltration, necrosis of alveolar walls andpulmonary edema. Immunohistochemistry staining revealed high quantitiesof viral antigen in the MVA-LUC control group (FIGS. 16D, H, L & P). Incontrast, viral antigen was not detected in lungs of mice that receivedMVA-H5M (FIGS. 16C, J, K & O). These data demonstrate the ability ofMVA-H5M to confer both broad and strong protection against multipleclades of avian influenza viruses.

The ability of the MVA-H5M construct to protect mice against seasonalinfluenza viruses was also evaluated. Vaccination followed the sameprotocol as with H5N1 viruses, except mice were challenged with A/PuertoRico/8/1934 (PR8; H1N1) and A/Aichi/2/1968 (H3N2). At four weekspost-vaccination, neutralizing Ab titers were below detectable levelsagainst both seasonal influenza viruses (FIG. 5B). Despite the lack ofdetectable neutralizing Ab, MVA-H5M conferred complete protectionagainst PR8 (H1N1) with no significant weight loss observed (FIGS. 13Hand K). In contrast, no protection was observed against A/Aichi/2/68(H₃N₂) challenge (FIGS. 13I and L). Regardless of strain, viralreplication was observed in the lungs of mice vaccinated with MVA-H5Mand challenged with seasonal influenza viruses; however, mice challengedwith PR8 had significantly lower viral lung titers than mice vaccinatedwith MVA-LUC controls (FIG. 13N). In contrast, vaccination with MVA-H5Mhad no effect on viral replication in the lungs of mice challenged withA/Aichi/2/68 (FIG. 13N).

Short- and Long-Term Immunity

To assess the ability of the MVA-H5M construct to confer both short- andlong-term immunity, groups of mice vaccinated with a single dose MVA-H5Mwere challenged at either 10 days or 6 months. MVA-H5M provided fullprotection against a lethal dose of A/HK/483/97, at both 10 days and 6months post-vaccination (FIGS. 14A and B). Additionally, neutralizingantibodies against A/HK/483/97 were detected at both 10 days and 6months post-vaccination (FIG. 14C). Microneutralization assays wereconducted using A/HK/483/97 because it is the most virulent strain amongthe four H5N1 strains used in the present study. Surprisingly, GMT Abtiters at 6 months were substantially higher than those detected at 4weeks post-vaccination (FIGS. 15B & 4C). In these animals, H5N1-specificIFN-γ CD4⁺ and CD8⁺ T cell responses were detected using flow cytometry.IFN-γ-releasing CD4⁺ and CD8⁺ T cells were found in MVA-H5M vaccinatedmice 5 months after dosing (2 weeks before challenged in long-termprotection study), indicating a long-term memory response (FIG. 14D).

Discussion

The rapid evolution of influenza viruses poses global health challengesnecessitating development of vaccines with broad cross-protectiveimmunity. Herein, the development of a broadly protective vaccine,MVA-H5M, based on a mosaic epitope approach, is described. The mosaicapproach minimizes genetic differences between selected vaccineantigenic sequences and circulating influenza strains while maximizingthe overall breadth of cross-protective immune responses. The presentresults demonstrated that a single dose of MVA expressing a mosaic H5hemaglutinin (MVA-H5M) provided broad protection against multiple H5N1viruses, including the highly pathogenic Egyptian strains, and also anH1 subtype virus (PR8). The MVA-H5M vaccine provided robust andprolonged protection against a lethal dose of highly pathogenic avianinfluenza as early as 10 days and as long as 6 months post vaccination.

In the past few years, commercially available vaccines have failed toinduce the expected level of protection against the currentlycirculating clade 2.2.1 in Egypt (Bahgat et al., 2009; Hafez et al.,2010). It is very important that an H5N1 influenza vaccine provide broadcross-clade protection against these 2.2.1 viruses particularly theA/Egypt/1/08 strain, because this strain possess one of the fourmutations that are necessary to sustain human to human transmission(Herft et al., 2012). The MVA-H5M vaccine showed complete protectionagainst this H5N1 strain in mice. The ability to provide completeprotection against H5N1 viruses with a single dose is also important forimplementation; societal acceptance of a single dose vaccine wouldlikely be higher than for a multi-dose vaccine, especially during apandemic.

Several plausible hypotheses exist for the exact mechanism by which themosaic vaccine confers broad protection against influenza viruschallenge. One possible explanation is that the 12-mer mosaic sequencecaptured more T-helper eptitopes, in which case the broad protectiveability of MVA-H5M likely results from greater epitope coverage for themosaic than for previous approaches (Santra et al., 2010 and FIG. 6).This could translate to a higher level of CD4⁺ T cells and broaderantibody responses than induced by wild-type sequence (MVA-HA). Thishypothesis is supported by the fact that MVA-H5M showed broaderIFNγ-CD4⁺ T cell epitope coverage and broader cross-clade neutralizingantibody responses (FIGS. 8 and 9). However, other immunological aspectsof the MVA-H5M vaccine still need to be further characterized. Forexample, data on CD8⁺ T cell responses, cytokine profiles, antibodyepitope coverage and mapping would all be necessary to fully understandthe mechanism responsible for protection.

A second mechanism that may explain the breadth of protection conferredby the MVA-H5M vaccine is that the mosaic approach maintains intactantigenic structure and presumably physiological function (Santra etal., 2010; Kaur et al., 2011). It has been previously reported that mostuniversal neutralizing antibodies are elicited by peptides in the stalkregions (Kaminski et al., 2011; Kaur et al., 2011). The MVA-H5M vaccinehas normal hemagglutination function and also is expressed as acleavable protein. Furthermore, the mosaic H5 might provide higheraccessibility to the stalk region and stimulate a more robustneutralizing antibody response against epitopes in the stalk region.Crystallography of expressed mosaic H5 would likely be required toreveal the actual structure of this protein and compare it to the knownstructure of H5 hemagglutinin.

The MVA-H5M provided sterilizing lung protection with no mortality andno morbidity against H5N1 viruses (FIG. 13). Moreover, no viral antigenswere detected in the lung after challenge (FIG. 14). These results arelikely due to high neutralizing antibodies (at least 1:32 end-pointtiter) (FIG. 5). Previous reports have demonstrated that a minimumneutralizing antibody concentration of 1:16 end-point titer issufficient to provide complete protection against H5N1 viruses (Howardet al., 2011). Although antibody mediated protection is suggested to bethe main contributor of protection in the present vaccine, T cells mayalso play a role.

It is currently unclear whether the use of a live viral vector such asMVA contributed to the increased cross-protection described herein. Itis possible that H5M expression by MVA induced high levels of crossreactive neutralizing antibodies as well as HA-specific IFN-γ-secretingCD4⁺ and CD8⁺ T cells. Specific CD4⁺ and CD8⁺ T cells that were inducedby MVA-H5M vaccine recognized different regions of diverse H5N1 peptides(FIG. 6), as well as recognizing specific conserved epitopes that hadbeen previously reported (Kuwano et al., 1991). The protective efficacyof the H5M antigen as a recombinant protein may require the use ofadjuvants and multiple doses to achieve desired protection.

The mosaic approach has been previously used for developing vaccinesagainst the highly variable HIV viruses, capturing potential CD8 T cellepitopes with a length of nine amino acids (Barouch et al., 2010;Fischer et al., 2007) while still maintaining normal protein structure.Because complete protection against influenza viruses is based primarilyon humoral immunity (Chiu et al., 2013; Niqueux et al., 2010), thealgorithm for epitopes of 12 amino acids was modified in order tocapture potential T helper cell epitopes (Gogolak et al., 2000;Ben-Yedidia and Amon, 2005) in order to target antibody producing plasmacell via T helper cells activation. This strategy may have facilitatedMVA-H5M achieving high neutralizing antibody with single dose (FIG. 5).

The vaccine elicited strong humoral responses against multiple H5N1viruses but no cross-neutralizing antibodies against seasonal influenzaviruses (H1N1 and H3N2). Despite the lack of neutralizing antibodiesagainst H1N1 PR8 virus, the MVA-H5M vaccine provided 100% protectionagainst PR8. This suggests a substantial role of cellular immuneresponses against PR8 virus, as shown in FIG. 9, likely because the H5Mprotein possesses some CLT epitopes of PR8 (Bui et al., 2007). Thisprotection can also be explained by the genetic relationship between theH5 and H1 hemagglutinin subtypes, as both belong to group 1 (Liu et al.,2009), and it elicits high amount of cross non-neutralizing antibodywhich then target and destroy H1N1 virus via antibody-dependent cellularcytotoxicity (ADCC) mechanism (Jegaskanda et al., 2013). However, theMVA-H5M vaccine did not protect immunized mice against influenzaA/Aichi/2168, which likely is due to antigenic differences as the H3belongs to group 2 hemagglutinin. Because the MVA vector can be designedto contain multiple inserts, future constructs will contain mosaics fromseveral hemagglutinin groups, including important seasonal (e.g H3s) andemerging (e.g., H7s) pathogens. Since this vaccine provides broadprotection and a long duration of immunity, utilizing an MVA vectorexpressing seasonal mosaics might diminish the need for annualvaccination.

The ability of MVA-H5M vaccine to confer broad protective immunityagainst various homologous strains as well as heterosubtypic strainsmakes the mosaic approach a very promising strategy to combat theantigenic diversity of influenza viruses. Taken together with codonoptimization of HA for high level of protein expression and using MVAvector as a backbone for cellular immunity activation, this approachpromises to increase the broad efficacy of influenza vaccinessubstantially. Should this and similar approaches prove effective forother viruses in other animal models, it could help reduce or eliminatethe need for annual seasonal influenza vaccine “updates,” as well asproviding a framework for a “pandemic preparedness” vaccine.

Example II

Samples were collected for a microneutralization test at 4 weekspost-immunization. FIG. 7 shows the antibody titers for three differentstrains of influenza virus in mice immunized with H5M/MVA, inactivatedvikeus (Baxter) or MVA alone. The antisera in immunized mice werereactive against three distinct viral clades (HPAI Clade 0, Clade 1, andClade 2 viruses) after a single dose. The presence of neutralizingantibodies at 4 weeks indicates that the H5M vaccine provides rapidimmunity and the immunity is higher than inactivated against thehomologous virus. Even recent consensus approaches that have tried tocontrol for the most diversity of input sequence have failed tosimultaneously elicit immune responses against all of these clades.

TABLE 1 Grp Constructs Route N = Challenge stains 1 MVA-H5M ID 8HK/483/97 2 Inactivated H5N1 SC 8 HK/483/97 3 MVA/LUC - control ID 5HK/483/97 4 MVA-H5M ID 8 MONG/244/05 5 Inactivated H5N1 SC 8 MONG/244/056 MVA/LUC - control ID 5 MONG/244/05 7 MVA-H5M ID 8 VN/1203/04 8Inactivated H5N1 SC 8 VN/1203/04 9 MVA/LUC - control ID 5 VN/1203/04

The vaccine protected 100% of the mice against all three challengestrains (FIG. 10). Thus, the MVA-H5M vaccine elicited robust and crossprotective immunity in mice against avian influenza strains clade 0,clade 1 and clade 2.2. MVA-H5M lowered weight loss, viral lung titersand prevented severe lung lesions (FIGS. 10-12).

Example 111

The genetic algorithm was used to generate other mosaic sequences. 4,809H1 sequences were used to generate 4 H1M sequences, e.g., using defaultparameters or modified parameters such as a different random seednumber. A characteristic residue in one set of those sequences (SEQ IDNos. 2 and 3) may be at position 125 (Ile), and a characteristic residuein the other set (SEQ ID Nos. 4 and 5) may be at one or more ofpositions 62 (Lys), 64 (Ile), 68 (Gln), 71 (Asn), 73 (Ser), 74 (Val), 86(Leu), 88-91 (IleSerLysGlu), 99-103 (LysProAsnProGlu), 111 (His), or 113(Ala), or corresponding positions (depending on the length of the signalpeptide).

2,931 H3 sequences were used to generate a H3M sequence (SEQ ID NO:7).

393 H2 sequences were used to generate a H2M sequence (SEQ ID NO:6). Acharacteristic residue in H2M may be at one or more of positions 24(Ala), 45 (Lys), 86 (Ser), 258 (Thr), 260 (Asn), or 261 (Leu), orcorresponding positions.

799 H7 sequences were used to generate a H7M sequence (SEQ ID NO:8). Acharacteristic residue in H7M may be at one or more of positions 91(Ser), 92 (Ser), 122 (Arg), 127 (Gly), 195 (Glu), 197 (Val), or 198(Ser), or corresponding positions.

927 H9 sequences were used to generate a H9M sequence (SEQ ID NO:9). Acharacteristic residue in H9M may be at one or more of positions 180(Gln), 215 (Glu) or 240 (Tyr), or corresponding positions.

212 H10 sequences were used to generate a H10M sequence (SEQ ID NO:10).A characteristic residue in H10M may be at position 77 (Val), or acorresponding position.

1,085 HA B sequences were used to generate a HBM sequence (SEQ IDNO:11). A characteristic residue in HBM may be at one or more ofpositions 86 (Met), 88 (Val), 90 (Thr), 91 (Thr), 95 (Lys), 96 (Ala), or161 (Val), or corresponding positions.

3,347 sequences were used generated a N1M sequence (SEQ ID NO:12), 4,444Mosaic sequences were used generated for N2 (SEQ ID NO:13) and 169sequences were used generated a N7 (SEQ ID NO:14). A characteristicresidue in N1M may be at one or more of positions 35 (Ala), 44-48(AsnHisThrGlyIle), 52 (Arg), 59 (Ser), 64 (His), 70 (Asn), 74-77(ValValAlaGly), 79-81 (AspLysThr), 99 (Ile), or 105 (Ser). Acharacteristic residue in N2M may be at one or more of positions 199(Lys) or 221 (Asn), or corresponding positions.

The approach may thus be employed with any subtype of influenza virus HAor NA, as well as influenza B virus, to generate one or more mosaicinfluenza antigens that are incorporated into a universal influenzavaccine which provides both domesticated animals and humans with themaximum possible protection against this devastating respiratorydisease. Moreover, polyvalent mosaic sequences based on HA and NA may beemployed to develop a universal influenza vaccine. The use of poxvirusas a delivery vehicle for the mosaic antigen facilitates immuneprotection because its replication triggers innate immunity as well as Tcell and B cell responses, and the poxvirus can be used in both birdsand humans; further, it can be given orally.

To modify the sequences described above, conserved regions may beidentified (see FIG. 12). For example, position coverage of mosaicsequences, e.g., for greater than or equal to 80% coverage for SEQ IDNO:1 (H5) includes residues 13-39, 60-75, 111-124, 300-314, 351-388,401-438, 451-477, 513-516, and 530-537; for SEQ ID NO:2 (H1) includes21-39, 88-89, 115-125, 284-287, 363-370, 436-439, 472-477, 528-532, and552-555; for SEQ ID NO:3 (H1) includes 21-24, 37-39, 363-369, 433-438,and 469-476; for SEQ ID NO:4 (H1) includes 21-39, 88-89, 115-125,363-370, 401-404, 422-423, 433-438, 469-477, and 528-532; for SEQ IDNO:5 (H1) includes 21-24, 37-39, 363-369, 469-476, 528-531, and 552-555;for SEQ ID NO:6 (H2) includes 25-33, 61-75, 104-108, 155-157, 253-271,287-289, 309-310, 325-339, 351-373, 386-389, 416-439, 457-458, 474-481,501-508, and 521-531; for SEQ ID NO:7 (H3) includes 24-29, 51-54,111-125, 301-303, 324-339, 351, 364-365, 378-379, 403-439, 451-454,470-483, 506-534, and 551-555; for SEQ ID NO:8 (H7) includes 131,151-153, 293-300, 351-376, 423-431, 464-474, and 511-512; for SEQ IDNO:9 (H9) includes 26-28, 51, 67, 108-109, 254-255, 337-339, 354-360,414-417, 430-439, and 451-457; for SEQ ID NO:10 (H10) includes 16,78-86, 101-114, 127-133, 151-154, 167-171, 201-234, 251-259, 301-310,351-389, 401-415, 419-439, 451-471, 488, 501-513, and 526-530; for SEQID NO:11 (HA B) includes 1-39, 101-119, 224-232, 251-254, 283-288,301-338, 351-388, 401-438, 451-481, 501-508, 521-538, 551-553, and570-574; for SEQ ID NO:12 (N1) includes 107-143, 174-176, 190-202,290-299, 398-404, 436-438, 474-484, 506-525, and 538-539; for SEQ IDNO:13 (N2) includes 1-4, 94-113, 156-160, 173-182, 222-237, 287-290,314-316, 415-419, and 438-451; for SEQ ID NO:14 (N7) includes 1-7,20-27, 106-114, 127-152, 264-272, 286-290, 338-344, 359-370, 392-402,417-435, and 456-460.

The conserved regions are those that are included in the mosaicsequences of the invention and may be substituted, e.g., up to 5%, 10%or 20%, relative to the corresponding sequences in any of SEQ ID Nos.1-14.

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What is claimed is:
 1. A recombinant nucleic acid molecule having a) anucleotide sequence encoding an immunogenic influenza virus HApolypeptide having one of SEQ ID NOs:1-11, a sequence with at least 99%amino acid sequence identity thereto, or a portion thereof, whichprovides cross-clade reactivity; b) a nucleotide sequence encoding animmunogenic H5 HA polypeptide having SEQ ID NO:1, or a sequence with atleast 95% amino acid sequence identity thereto or an immunogenic portionthereof, wherein the HA has Ile at position 87, Thr at position 172, Valat position 226 or Thr at position 279, or a combination thereof; c) anucleotide sequence encoding an immunogenic H1 HA polypeptide having SEQID NO:2, 3, 4, or 5, a sequence with at least 95% amino acid sequenceidentity thereto or an immunogenic portion thereof, wherein the HA i)has Arg at position 206, Leu at position 432, or Val at position 434, ora combination hereof; ii) has Ile at position 125 and Val at position564; or iii) has Lys at position 62, Ile at position 64, Gln at position68, Asn at position 71, Ser at position 73, Val at position 74, Leu atposition 86, Ile at position 88, Ser at position 89, Lys at position 90,Glu at position 91, Lys at position 99, Pro at position 100, Asn atposition 101, Pro at position 102, Glu at position 103, His at position111, or Ala at position 113, or a combination thereof; d) a nucleotidesequence encoding an immunogenic H2 HA polypeptide having SEQ ID NO:6, asequence with at least 95% amino acid sequence identity thereto or animmunogenic portion thereof, wherein the HA has Ala at position 24, Lysat position 45, Ser at position 87, Thr at position 258, Asn at position260, or Leu at position 261, or a combination thereof; e) a nucleotidesequence encoding an immunogenic H7 HA polypeptide having SEQ ID NO:8, asequence with at least 95% amino acid sequence identity thereto or animmunogenic portion thereof, wherein the HA has Ser at position 91, Serat position 92, Arg at position 122, Gly at position 127, Glu atposition 195, Val at position 197, or Ser at position 198, or acombination thereof; f) a nucleotide sequence encoding an immunogenic H9HA polypeptide having SEQ ID NO:9, a sequence with at least 95% aminoacid sequence identity thereto or an immunogenic portion thereof,wherein the HA has Gln at position 180, Glu at position 215, or Tyr atposition 240, or a combination thereof; g) a nucleotide sequenceencoding an influenza B HA polypeptide having SEQ ID NO:11, a sequencewith at least 95% amino acid sequence identity thereto or an immunogenicportion thereof, wherein the HA has Met at position 86, Val at position88, Thr at position 90, Thr at position 91, Lys at position 95, Ala atposition 96, or Val at position 161, or a combination thereof; h) anucleotide sequence encoding a H3 HA polypeptide having SEQ ID NO:7; i)a nucleotide sequence encoding a H10 HA polypeptide having having SEQ IDNO:10; j) a nucleotide sequence encoding an immunogenic influenza virusNA polypeptide having one of SEQ ID NOs:12-14; k) a sequence with atleast 95% amino acid sequence identity to SEQ ID NO:12 having Ala atposition 35, Ser at position 42, Asn at position 44, His at position 45,Thr at position 46, Gly at position 47, Ile at position 48, Arg atposition 52, Ser at position 59, His at position 64, Asn at position 70,Val at position 74, Val at position 75, Ala at position 76, Gly atposition 77, Asp at position 79, Lys at position 80, Thr at position 81,Ile at position 99, or Ser at position 105, or a combination thereof; I)a sequence with at least 99% amino acid sequence identity to SEQ IDNO:13 having Lys at position 199, Asn at position 221, or Gln atposition 433, or a combination thereof; or m) a sequence with at least99% amino acid sequence identity to SEQ ID NO:14 having Ile at position353; or an immunogenic portion thereof.
 2. The recombinant nucleic acidmolecule of claim 1 wherein the nucleotide sequence is linked to apromoter operable in avian or mammalian cells.
 3. A vaccine comprising arecombinant virus, the genome of which comprises at least one expressioncassette having a promoter operably linked to a heterologous openreading frame comprising a nucleotide sequence for an influenza viruspolypeptide having one of SEQ ID NOs: 1-14, a polypeptide with at least95% amino acid sequence thereto or an immunogenic portion thereof, or acombination thereof, which provides cross-clade reactivity.
 4. Thevaccine of claim 3 further comprising an adjuvant.
 5. The vaccine ofclaim 3 further comprising a different virus.
 6. The vaccine of claim 3further comprising a pharmaceutically acceptable carrier.
 7. The vaccineof claim 3 wherein the carrier is suitable for intranasal orintramuscular administration.
 8. The vaccine of claim 3 which is infreeze-dried form.
 9. The vaccine of claim 3 which is adapted formucosal, intramuscular or intradermal delivery.
 10. A method to prevent,inhibit or treat influenza virus infection comprising administering toan animal or an egg thereof, a composition comprising an amount of atleast one recombinant virus, the genome of which comprises at least oneexpression cassette having a promoter operably linked to the recombinantnucleic acid molecule of claim 1, effective to induce an adaptive immuneresponse to influenza virus.
 11. The method of claim 10 wherein theanimal is an avian or a mammal.
 12. The method of claim 10 wherein thecomposition is intradermally administered.
 13. The method of claim 10wherein the composition is intramuscularly administered.
 14. The methodof claim 10 wherein the composition is mucosally administered.
 15. Themethod of claim 10 wherein the effective amount is administered in morethan one dose.
 16. The method of claim 10 wherein the compositionfurther comprises an adjuvant.
 17. The method of claim 10 wherein thecomposition is parenterally administered.
 18. The method of claim 10wherein the composition is administered intranasally.
 19. The method ofclaim 10 where the composition is administered orally.
 20. The method ofclaim 10 wherein the amount prevents or inhibits influenza virusinfection across two or more clades.